Critical Reviews in Analytical Chemistry

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/batc20

Diuretics in Different Samples: Update on the

Pretreatment and Analysis Techniques

Ya-jie Liu, Yu Bian, Yuan Zhang, Yi-xin Zhang, Ai Ren, Shu-han Lin, Xue-song
Feng & Xin-yuan Zhang

To cite this article: Ya-jie Liu, Yu Bian, Yuan Zhang, Yi-xin Zhang, Ai Ren, Shu-han Lin,
Xue-song Feng & Xin-yuan Zhang (2023): Diuretics in Different Samples: Update on the
Pretreatment and Analysis Techniques, Critical Reviews in Analytical Chemistry, DOI:
10.1080/10408347.2023.2202260

To link to this article: https://doi.org/10.1080/10408347.2023.2202260

View supplementary material

Published online: 02 May 2023.

Submit your article to this journal

Article views: 111

View related articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=batc20
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY
https://doi.org/10.1080/10408347.2023.2202260

Diuretics in Different Samples: Update on the Pretreatment and Analysis

Techniques
Ya-jie Liua
, Yu Biana
, Yuan Zhanga
, Yi-xin Zhanga, Ai Rena, Shu-han Linb, Xue-song Fenga , and Xin-yuan
Zhangc
a
School of Pharmacy, China Medical University, Shenyang, China; bSchool of Food Science and Engineering, Dalian Ocean University, Dalian,
China; cSchool of Forensic Medicine, China Medical University, Shenyang, China

ABSTRACT KEYWORDS
Diuretics are drugs that promote the excretion of water and electrolytes in the body and produce Diuretics; pretreatment;
diuretic effects. Clinically, they are often used in the treatment of edema caused by various rea- determination; quality
sons and hypertension. In sports, diuretics are banned by the World Anti-Doping Agency (WADA). control; banned drugs
Therefore, in order to monitor blood drug concentration, identify drug quality and maintain the
fairness of sports competition, accurate, rapid, highly selective and sensitive detection methods
are essential. This review provides a comprehensive summary of the pretreatment and detection
of diuretics in various samples since 2015. Commonly used techniques to extract diuretics include
liquid-liquid extraction, liquid-phase microextraction, solid-phase extraction, solid-phase microex-
traction, among others. Determination methods include methods based on liquid chromatography,
fluorescent spectroscopy, electrochemical sensor method, capillary electrophoresis and so on. The
advantages and disadvantages of various pretreatment and analytical techniques are elaborated.
In addition, future development prospects of these techniques are discussed.
HIGHLIGHTS
 Pretreatment and determination methods of diuretics in diverse samples are reviewed.
 Applications of novel materials and technologies for SPE and sensors are highlighted.
 Pros and cons of recent pretreatment and analysis techniques used for diuretics are discussed.
 Applications of high-resolution mass spectrometry are described in detail.

1. Introduction canrenone (CAN), chlortalidone (CHL), etacrynic acid, fur-

Diuretics are the part of treatment strategies for patients
with fluid retention such as nephrotic syndrome, chronic
kidney disease, heart failure and cirrhosis,[1] which may lead
to lower blood pressures and dehydration. Based on diverse
action mechanisms,[2] diuretics can have six categories: loop
diuretics, thiazides and thiazide like diuretics, potassium
preserving diuretics, osmotic diuretics, carbonic anhydrase
inhibitors and vasopressin V2 receptor antagonists. The six
drugs and their action mechanisms, indications and side
effects are summarized in Table S1. Diuretics are used in
treating multiple diseases. The drugs content is important
for patients’ health evaluations. It is thus imperative to
detect and quantify the diuretics content in commercial for-
mulations and the drugs concentrations during treatments.[3]
The detection of diuretics in athletes’ urine has become
important as taking diuretics can mask the existence of
stimulants and thus invalidate the drug tests of athletes.[4]
Diuretics and masking agents are therefore included in the
World Anti-Doping Code international standard prohibited
list 2022. It includes but not limited to mannitol (MAN),
acetazolamide (ACE), amiloride (AMI), bumetanide,
osemide (FUR), indapamide (IND), metolazone, spironolac-
tone (SPI), thiazides, triamterene (TRI), and vaptans.
Developing quick, accurate, sensitive, and high throughput
analysis methods for diuretics is vital to determine the kinds
of diuretics and their distinct chemical structures.
Browsing the literature since 2015 could find only one
mini review on detection of diuretic doping. Nan et al.[5]
elaborated the progress in electrochemical and electrophor-
etic determination of diuretics doped in stimulants from
athletes’ urine, blood, saliva and hair. While they focused on
limited detection techniques are not enough as different test-
ing purposes are required such as screening, more sensitive
and selective. Besides, in recent years, analysis techniques
like chromatography, sensors, spectroscopy, and others have
been developed prosperously for determining diuretics in
diverse matrices with regard to different testing require-
ments. What’s more, pretreatment methods with novel
materials and devices for diuretics which is a critical process
before determination has been widely utilized and developed
rapidly. Nevertheless, till now, no comprehensive review has

CONTACT Xue-song Feng xsfeng@cmu.edu.cn School of Pharmacy, China Medical University, Shenyang 110122, China; Xin-yuan Zhang
20092002@cmu.edu.cn School of Forensic Medicine, China Medical University, Shenyang 110122, China

The author contributed equally to the work.
Supplemental data for this article can be accessed online at https://doi.org/10.1080/10408347.2023.2202260.
ß 2023 Taylor & Francis Group, LLC
2 Y.-J. LIU ET AL.
been published on the pretreatment and determination
methods for diuretics.
Accordingly, an up-to-date and comprehensive overview
of the latest developments for diuretics in different matrices
regarding both the pretreatment and detection methods is
urgently needed. The pretreatment and detection methods
of six diuretics with different chemical structures in diverse
samples are summarized in detail. Commonly used pretreat-
ments include protein precipitation, solid-phase extraction,
solid-phase microextraction, liquid-phase microextraction,
and others. The detections of diuretics in diverse matrices
are made through chromatography, spectrophotometry, vol-
tammetry, sensors, and others. The pros and cons of devel-
oped methods are provided and upcoming method
development trends are prospected. Moreover, emphasis is
placed on the extraction methods for diuretics based on
novel materials and high-resolution mass spectrometric
(HRMS) methods.
2. Sample pretreatment methods
Sample pretreatment refers to the gas, liquid or solid sam-
ples treatment by various methods and technologies to
achieve following purposes: (1) concentrate the tested com-
ponents and improve method sensitivity; (2) remove matrix
and interfering substances; (3) derivatization and other
Figure 1. Sample preparation methods for diuretics in diverse matrices.
reactions to increase the method selectivity; and (4) protect
subsequent analytical instruments and test systems. Owing
to the diversity of matrices, and low level of diuretics, the
applications of suitable pretreatment techniques are in
necessity for getting accurate, sensitive and satisfactory
results (Figure 1). The pretreatment methods of diuretics
reported between 2015 and 2022 have been described in the
following sections, and some reports were listed in Table 1.
2.1 Traditional pretreatment methods for diuretics
PPT is a prevailing pretreatment method for analyzing diu-
retics in biological samples. Protein precipitants like organic
solvents (acetonitrile (ACN))[41] and some acids (trichloro
acetic acid)[42] are used to precipitate proteins. It is a simple,
fast, and convenient method to achieve high recovery.[41]
This method however removes only proteins and endogen-
ous impurities are not discarded. Analyte signals are thus
suppressed and fail to achieve satisfactory limit of detection
(LOD). Moreover, the long-term detections may affect the
life of chromatographic column and instrument.[13] UAE
has been used as an alternative method for processing vari-
ous types of samples without obvious signal suppression.[43]
It shortens the extraction time and has good extraction effi-
ciency achieved by combining the effects of cavitation, vibra-
tion, crushing, and stirring produced by ultrasonic waves.[44]
Table 1. Different extraction methods for diuretics in diverse matrix.
Pretreatment
Matrix Analytes methods Pretreatment conditions Performance Ref.
[6]
Human plasma IND PPT Sample volume: 100 lL Extraction time: 11 min
Extractant: 200 lL ACN Recovery (%): 94.5–98.5
RSD (%): <15
[7]
Human plasma TOL and its metabolites PPT Sample volume: 100 lL Recovery (%): 99.1–102.5
Extractant: 600 lL ACN RSD (%): 3.0–11.1
[8]
Human plasma TOR PPT Sample volume: 200 lL Extraction time: 13 min
Extractant: 2.0 mL ethyl acetate with Recovery (%): 84.20–86.47
20 lL 10% HCl RSD (%): 2.63–8.67
[9]
Tablets HCT UAE Sample volume: 795 mg Extraction time: 25 min
Extractant: 20 mL phosphoric acid Recovery (%): 92–104
solution: ACN: MeOH (40:30:30, v/v/v) RSD (%): 0.3–0.7
[10]
Human plasma HCT LLE Sample volume: 300 lL Extraction time: 15 min
Extractant: 2.5 mL diethyl ether: Recovery (%): 82.83
dichloromethane (70:30, v/v) RSD (%): 4.34
[11]
Human plasma HCT LLE Sample volume: 200 lL Extraction time: 20 min
Extractant: 100 lL 0.5 M HCl and 1 mL Recovery (%): 73.54–79.22
methyl tertiary butyl ether: RSD (%): 1.4–7.2
dichloromethane (85:15, v/v)
[12]
Rabbit plasma TOL LLE Sample volume: 50 lL Extraction time: 10 min
Extractant: 3 mL methyl tertiary butyl Recovery (%): 87.10
ether: dichloromethane (80:20, v/v) RSD (%): <13.19
[13]
Human plasma CHL Two-step LLE Sample volume: 50 lL Extraction time: 30 min
Extractant: 50 mL 2% formic acid and Recovery (%): 84.20–86.47
2.5 mL n-hexane-ethyl acetate (50:50, RSD (%): <13.19
v/v), repeat once
[14]
Water matrix FUR DLLME-SFO Sample volume: 10 mL Extraction time: 10 min
Extractant:100 lL 1-dodecanol Recovery (%): 80–120
Disperser solvent:500 lL MeOH RSD (%): 8.0–9.0
[15]
Human urine HCT and TRI Two-phase and Sample volume: 24 mL Extraction time: 90 min
three-phase HF- Extractant: 25 lL 1-decanol (containing Recovery (%): 89.5-98.0
LPME aliquat-336); 1-decanol (pH ¼ 1.0) RSD (%): 5.9–12.2
Sample pH: 12; 3.0 mol/L NaOH PF: 128 and 239
Polypropylene hollow fiber
Reused times: disposal
[16]
Human urine ACE, bendroflumethiazide, MWCNTs-HF-LPME Sample volume: 7.5 mL Extraction time: 34 min
CTA, HCT, Extractant: 25 lL 10 mM ammonia Desorption time: 5 min
hydroflumethiazide, solution Recovery (%): 89–96
trichlormethiazide and Organic phase: n-octanol RSD (%): <6
althiazide Immobilized MWCNTs hollow fiber EF: 135–1587
Reused times: 50 (recovery >85%)
[17]
Human urine TRI MIPs-SPE Sample volume: 1 mL Extraction time: 12 min
Sorbent: 2,4-diamino-6-methyl-1,3,5- Recovery (%): 80.9–89.8
triazine-MIPs (100 mg) RSD (%):2.8–4.1
Precondition: 3.0 mL MeOH and 3.0 mL IF: 2.92
water
Wash: 3.0 mL ACN
Elution: 5 mL mixture MeOH: ammonia
(95:5, v/v)
[18]
Human urine IND MIPs-SPE Sample volume: 90.0 mL Extraction time: 180 min
Sorbent: MIPs (50 mg) Recovery (%): 80.1–81.2
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY

Precondition: 2  0.5 mL loading RSD (%): 0.17–1.4

solvent (ACN or pH 5.2 distilled water)
(continued)
3
 4

Table 1. Continued.
Pretreatment
Matrix Analytes methods Pretreatment conditions Performance Ref.
Wash: 4  0.5 mL ACN Preconcentration factor: 16.3
Elution: 8  0.5 mL MeOH IF: 9.3
[19]
Human urine Azosemide, buthiazide, MSPE Sample volume: 10 mL Extraction time: 24.38 min
brinzolamide and Sorbent: Fe3O4@UiO-66-OH (10 mg) Desorption time: 10 min
Y.-J. LIU ET AL.

dorzolamide Desorption: 2 mL ACN Recovery (%): 90.3–97.7

RSD (%):1.78-4.07
EF: 93–97
[20]
Human urine FUR on-line mSPE Sample volume: 1 mL Extraction time: 18 min
Sorbent: MNPs-polybutylene Desorption time: 120 s
terephthalate (21 mg) Recovery (%): 85.0
Elution: ACN and filtrated double- RSD (%):7.8
distilled water with 0.1% H3PO4
(1:1, v/v)
[21]
Human urine HCT d-SPE Sample volume: 10.0 mL Extraction time: 25 min
Sorbent: Fe3O4@MIPs (50.0 mg) Desorption time: 15 min
Wash: 3.0 mL deionized water Recovery (%):90.7–110.0
Elution: 2.0 mL ethanol: acetone: RSD (%): 0.8–6.6
ammonium hydroxide (45:45:10)
solution
[22]
Human urine FUR BAlE Sample volume: 25 mL Extraction time: 16 h
Sorbent: Polymers P5 (0.9 mg) Desorption time: 1 h
Desorption: MeOH: ACN (1:1, v/v) Recovery (%): 91.4
RSD (%): 5.4
EF: 250
[23]
Human urine FUR and SPI SBME Sample volume: 30 mL Extraction time: 50 min
Sorbent: C18 sorbents Desorption time: 10 min
Desorption: 250 mL MeOH Recovery (%): 14.6–37.4
RSD (%):1.3–3.1
[24]
Human plasma IND SPE Sample volume: 75 mL Recovery (%): 89.4–90.5
Sorbent: Waters Oasis HLB 96-well RSD (%): 2.2–6.5
plate (10 mg) EF: 93-97
Precondition: 800 mL MeOH and 800 mL
purified water
Wash: 2  800 mL purified water
Elution: 2  400 mL MeOH
[25]
Human plasma HCT SPE Sample volume: 500 lL Recovery (%): 74.8–77.6
Sorbent: DVB-HL extraction cartridges RSD (%): 3.57–5.17
(30 mg)
Precondition: 1.0 mL MeOH followed by
1.0 mL 0.1 % acetic acid in water
Wash: 2  1.0 mL 0.1 % acetic acid in
water
Elution: 750 mL 2 % ammonia solution
in MeOH
[26]
Human plasma HCT SPE Sample volume: 100 lL Extraction time: 2 min
Sorbent: Oasis HLB cartridges Recovery (%): 88.5–96.3
Precondition: 1.0 mL MeOH followed by
1.0 mL water
Wash: MeOH in water and 1.0 mL 5 mM
ammonium formate
Elution: 500 mL ACN: water (90:10, v/v)
[27]
Human plasma HCT SPE
 Sample volume: 100 lL Extraction time: 1 min
Sorbent: Oasis HLB extraction cartridges Recovery (%): 96.6–103.1
(1 mL, 30 mg) RSD (%): 0.91–4.57
Precondition: 1.0 mL MeOH followed by
1.0 mL 5.0 mM ammonium formate (pH
3.0)
Wash: 1.0 mL 5.0 mM ammonium
formate solution followed by 1 mL
water
Elution: 0.5 mL mixture ACN and
5.0 mM ammonium formate (pH 4.5,
adjusted with 0.1% formic acid)
(85:15, v/v)
[28]
Human plasma AMI and HCT SPE Sample volume: 250 mL Extraction time: 1 min
Sorbent: Phenomenex StrataTM-X Recovery (%): 85.7–99.4
cartridges (30 mg, 1 mL) RSD (%): 0.66–6.24
Precondition: 1.0 mL MeOH followed by
1.0 mL 10 mM ammonium formate
solution
Wash: 1.0 mL 10 mM ammonium
formate in water, followed by 1.0 mL
10% (v/v) MeOH in water
Elution: 500 mL ACN and 4.0 mM
ammonium formate, pH 4.0 (80:20, v/v)
mixture
[29]
Human plasma HCT SPE Sample volume: 150 lL Extraction time: 2 min
Sorbent: Phenomenex Strata-X Recovery (%): 87.95–94.70
cartridges (30 mg, 1 mL) RSD (%): 3.03
Precondition: 1 mL MeOH followed by
1 mL water
Wash: 1 mL 5% MeOH in water
followed by 1 mL Milli-Q water
Elution: 0.5 mL elution solution (0.2%
formic acid in water: MeOH (20:80, v/v)
[30]
Human plasma IND SPE Sample volume: 250 lL Extraction time: 10 min
Sorbent: Oasis HLB cartridges (30 mg, Recovery (%): 96.64–98.64
1 mL) RSD (%):0.63–1.14
Precondition: 1 mL ACN, 1 mL MeOH,
and 1 mL water: ACN (95:5, v/v)
Wash: 5  1 mL MeOH: water (10: 90,
v/v)
Elution: 1 mL ACN
[31]
Human plasma HCT SPE Sample volume: 475 mL Extraction time: 1 min
Sorbent: Waters Oasis HLB cartridges Recovery (%): 91.76–96.51
(1 cc, 30 mg) RSD (%):0.67–1.33
Precondition: 1.0 mL MeOH followed by
1.0 mL 2.0 mM ammonium formate
Wash: 1.0 mL 2.0 mM ammonium
formate solution followed by 1.0 mL, 5
% MeOH in water
Elution: 500 mL MeOH
[32]
Human plasma TOL and its metabolites SPE Sample volume: 0.1 mL Recovery (%): 90.2–99.2
Sorbent: OASIS HLB 1 cc RSD (%): 0.7–13.1
Precondition: 1 mL MeOH followed by
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY

1 mL purified water
(continued)
5
 6

Table 1. Continued.
Pretreatment
Matrix Analytes methods Pretreatment conditions Performance Ref.
Wash: 1 mL 15% MeOH in water
Elution: 1 mL 60% ACN in water
[33]
Human plasma AMI, FUR and HCT SPE Sample volume: 500 lL Recovery (%): 51.0–95.0
Sorbent: Strong cation mixed mode
Y.-J. LIU ET AL.

Strata X-C cartridge

Precondition: 1 mL MeOH and 1 mL a
0.1% formic acid solution
Wash: 500 lL 0.1% formic acid solution
containing 3.4% of MeOH
Elution: 2.5 mL a methanolic solution of
ammonium hydroxide (2.2%)
[34]
Human plasma HCT, FUR and SPI SPMMTE Sample volume: 200 mL Extraction time: 30 min
Sorbent: Iron nanocomposite sorbent Desorption time: 30 min
(5.0 mg) Recovery (%): 85.2–90.4
Elution: MeOH with 0.1% acetic acid RSD (%): 0.98–1.6
[35]
Rat plasma HCT and TRI d-SPE Sample volume: 200 mL Extraction time: 8 min
Sorbent: 50 mg MAX and C18 Recovery (%): 82.1–89.9
Elution: 2 mL 5 % ammonia-ACN RSD (%): 1.8–3.8
[36]
Human serum TRI MIPs-SPE Sample volume: 1 mL Recovery (%): 86.8–94.8
Sorbent: DMT-MIPs (200 mg) RSD (%): 3.6–3.9
Precondition: 3.0 mL MeOH and 3.0 mL IF: 7.11
water
Wash: 2 mL MeCN
Elution: 5 mL MeOH: ammonia
(95:5, v/v)
[37]
Human serum and ACE d-SPE Sample volume: 1 mL Extraction time: 30 min
plasma Sorbent: MIPs (5 mg) Desorption time: 30 min
Elution: Tris HCl buffer (pH 8.5) Recovery (%): 85.0–96.0
RSD (%): < 5.8
IF: 2.8
[38]
Dietary Supplements CHL, HCT, IND and TAT UAE, d-SPE Sample volume: 2 mL Extraction time: 2 min
Sorbent: MWCNTs (20.0 mg) Recovery (%): 72.6–94.9
Wash: 2.00 mL MeOH: water (3:7, v/v) RSD (%): 1.48–9.14
Elution: 5.00 mL MeOH: 4% phosphoric
acid (96:4, v/v)
[39]
Aqueous samples FUR and HCT SPME Sample volume: 8 mL Extraction time: 60 min
Sorbent: PS/PEGDA fiber Recovery (%): 95.4–100.2
Desorption: 150 lL MeOH RSD (%): 1.6–7.8
EF: 409–878
[40]
Animal-derived HCT, SPI, CAN, CTZ QuEChERS PSA/C18/MgSO4 Sample volume: 15 mL Extraction time: 13 min
foods and ACE (100/100/600 mg) Extractant: 15 mL ACN Recovery (%): 72.1–118.1
RSD (%): 1.15–17.3

The operation is simple and components are easy to separ-
ate and purify. Huerta et al.[45] applied UAE for extracting
hydrochlorothiazide (HCT) from freshwater invertebrates
with mean recovery of 85% HCT in 6 min and consuming
3 mL methanol (MeOH). While it has demerits of high
noise, expensive equipment and large organic solvents con-
sumption, and not suits for complex matrices such as bio-
logical samples, which has promoted the development of
other diverse forms of extraction technology.
2.2. LLE
LLE utilizes different solubility or distribution ratio of com-
ponents in two immiscible solvents for achieving extraction
and purification. Compared with PPT, LLE consumes less
organic solvents with higher selectivity and less interference
by the matrix.[46] Different types of diuretics can be
extracted by LLE. Zhang et al.[8] extracted torasemide
(TOR) from human plasma using tolbutamide (10 lL) as IS,
ethyl acetate (2.0 mL) as extractant and 10% hydrochloric
acid solution (20 lL) as additive. Recovery of 84.20–86.47%
in 13 min was achieved from 200 lL sample. As a mature
technique, LLE has been improved continuously to realize
diverse demands of extraction such as more selective and
environmental-friendly.
Double extraction, also known as two-stage forward
extraction, has been developed for achieving higher selectiv-
ity and lower matrix effect (ME) in samples containing mul-
tiple analytes of diverse physicochemical properties. This
method refers to the LLE of matrix containing water sam-
ples with highly non-polar solvents first, and further extrac-
tion with medium non-polar solvents.[46] Elkady et al.[47]
simultaneously quantified HCT, olmesartan and amlodipine
from human plasma samples. Sequential double LLE was
selected as the optimum method owing to different pKa and
log P of three analytes. In the first extraction, basified
(0.05% ammonia) extraction mixture of diethyl ether and
dichloromethane (75:25, v/v) was added to samples solution
for extracting basic analytes (olmesartan and amlodipine). In
second round, acidic analytes (HCT) were extracted by the
acidified (0.3% acetic acid) mixture of diethyl ether and
dichloromethane (75:25, v/v). The extraction recoveries of
83%, 75%, and 63% for HCT, amlodipine and olmesartan
were obtained. Besides, the IS normalized matrix factor of
HCT, amlodipine and olmesartan were 4%, 3%, and 1%,
respectively, demonstrating the little matrix influence.
A modified LLE called cloud point extraction (CPE) has
emerged in recent years for avoiding the harmful organic
solvents. It is based on the solubility and cloud point of
aqueous solution of neutral surfactant micelles with no
harm to people. Experimental parameters are changed to
initiate the phase separation, and separate hydrophobic sub-
stances from hydrophilic without particular laboratory
equipment.[48] Mahdi et al.[49] used CPE to extract ACE
from pharmaceutical preparations. The recoveries of ACE
were from 98.0% to 100.1% by using Triton-x114 (0.5 mL)
as surfactant after 10 min of hot water bath (55  C) and
10 min of centrifugation. Besides, the enrichment factor (EF)
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 7
and preconcentration factor (PF) were 100 and 6.30, respect-
ively, which solve the problem of low EF in traditional LLE.
2.3. LPME
Though LLE can obtain high extraction efficiency, it con-
sumes large quantities of organic reagents. LPME was intro-
duced in 1990s.[50] Compared with LLE, LPME is simpler,
faster and uses microliter quantities of solvents. LPME such
as hollow fiber liquid-phase microextraction (HF-LPME)
and dispersive liquid–liquid microextraction (DLLME) are
popular, efficient, and environmental-friendly.
2.3.1. HF-LPME
The process of HF-LPME refers to the extraction of analytes
from donor phase to acceptor phase in hollow fiber lumen
via the organic solvent present in the pores of porous hollow
fiber. Two-phase HF-LPME system is created because of the
same organic solvent existing in fiber cavity and fiber pore.
When acceptor phase is aqueous, the organic phase in fiber
pores acts as barrier between acceptor and donor phases,
thus forming three-phase HF-LPME system.[51] The three-
phase system is applicable to ionizable acidic or alkaline
compounds. HF-LPME is simple, low-cost, and environment
friendly with high extraction efficiency. Ahmad Panahi
et al.[15] used two-phase and three-phase HF-LPME for
extracting HCT and TRI from urine samples respectively.
Polypropylene microporous hollow-fiber membrane was
selected as organic solvent carrier. For two-phase mode, the
optimal conditions were: organic solvent: 2.0% (w/v) ali-
quat-336 in 1-octanol, sample pH: 12, extraction time:
90 min. For three-phase mode, they were 1-decanol (pH ¼
1.0), 3.0 mol/L NaOH, 90 min, respectively and 2.0 mol/L
NaCl was added to sample solution to increase the distribu-
tion of analytes in 1-decanol. PFs for TRI and HCT were
239 and 128, and recoveries from 89.5  98.0%. To further
improve the extraction efficiency of HF-LPME for diuretics,
novel materials have been introduced.
Multi-walled carbon nanotubes (MWCNTs) are promis-
ing sorbents for diverse extraction techniques because of
their high surface area and unique tubular structure.[52]
They possess the inherent nature of carbon materials. In
addition, they also have the stitchability of textile fibers,
electrical and thermal conductivity of metals, machinability
of polymers and heat and corrosion resistance of ceram-
ics.[53] MWCNTs are thus excellent sorption materials for
variety of organic analytes due to unique physical and chem-
ical characteristics. Ho et al.[16] used three-phase immobi-
lized MWCNTs HF-LPME for extracting ACE,
bendroflumethiazide, CTA, HCT, hydroflumethiazide, tri-
chlormethiazide and althiazide from urine. 10 mM ammonia
solution (25 lL) was selected as extractant and n-octanol as
organic phase. EFs of these diuretics were between 135 and
1587 and recoveries from 89% to 96%, demonstrating super-
iority enrichment and extraction performance of this
method.
8 Y.-J. LIU ET AL.
Figure 2. The schematics of DLLME (A), MSPE (B) and BAlE (C).
2.3.2. DLLME
DLLME is based on ternary component solvent system of
sample solution, extractant and dispersant. Figure 2A shows
the common procedure of traditional DLLME. DLLME has
been prioritized because of high EFs, recovery, simplicity,
low cost, rapidity, and environment-friendliness.[54] Han
et al.[55] used DLLME to extract ethacrynic acid and FUR
from human urine. Using dichloromethane (50 lL) as
extractant and ACN (1000 lL) as dispersant, the EFs of two
diuretics were 89.9 and 103, respectively, and recoveries
84.6–96.0% after 5 min. Favorable extraction performance of
DLLME makes it widely used and attracts much attention of
researchers to improve it to get better results.
The dispersants used to separate extracts by centrifugation
include chlorinated solvents with density greater than water.
They are toxic and harmful to operators and environment,
and in need of being avoided. In 2007, DLLME with the
solidification of floating organic droplet (SFO) was introduced
where the extractant was less toxic and less dense than water
with melting point between 10 and 30  C.[56] Marube et al.[14]
used DLLME-SFO for extracting FUR from water matrix. 1-
dodecanol (100 lL) and MeOH (500 lL) were the extractant
and dispersant respectively. The recoveries of FUR were 80–
120% after 10 min. Satisfactory results exhibited that DLLME-
SFO had the probability to be a green alternative method to
extract diuretics from biological samples.
Due to the introduction of DLLME, emulsion-based
LPME has been developed to provide larger contact area for
improving the extraction efficiency and reduce extraction
time.[57] Regueiro et al.[58] proposed ultrasound-assisted
emulsification microextraction (USAEME) that combined
UAE to emulsion-based LPME for better extraction and
reducing the time. Ramkumar et al.[59] applied low-density
solvent (1-undecanol (40 lL)) instead of toxic organic solv-
ent (chlorinated solvents) with density higher than water as
extractant in USAEME for extracting IND from urine.
Recoveries of 80–119% were attained after 5 min, which
greatly shortened the extraction time.
2.4. SPE
Based on the theory of liquid-solid chromatography, SPE
enriches, separates and purifies samples by means of select-
ive adsorption and elution.[46] The main steps are as follows:
(i) conditioning of sorbents, (ii) loading samples, (iii) wash-
ing, and (iv) elution. Compared with LLE and SPE has more
advantages such as easier automation, effective separation of
analytes, less consumption of organic solvents and higher
recovery.[24] Some reports on SPE for extraction of diuretics
in diverse matrices were summarized in Table 1, and its
developments based on novel materials and new forms in
recent years are described below.
2.4.1. From traditional to novel materials for SPE
In SPE, sorbent material is the most significant. SPE can be
divided into four groups based on the differences in sorbents

and adsorption mechanisms: normal-phase SPE using polar
sorbents (alumina, diatomite, and silica gel, etc.) for extracting
polar compounds in non-polar solvents; reverse-phase (RP)
SPE with non-polar sorbents (C18, C8, etc.) for weakly polar
or non-polar analytes in polar matrix; ion-exchange SPE using
charged ion exchange resin (polyphenylene, diene, etc.) as
adsorbent to extract charged target compounds; and mixed
SPE (Oasis HLB, etc.) combined with multiple extraction
modes for separating complex matrix.[60]
Since 2015, Oasis HLB as SPE method[25–27] is being fre-
quently used in pretreating diuretic components from vari-
ous samples. It has two retention mechanisms,
hydrophilicity to retain polar drugs, and lipophilicity for
non-polar drugs. The acidic and alkaline drugs have higher
purification selectivity. Mahrouse et al.[61] applied SPE with
Oasis HLB for extracting antihypertensive drugs including
HCT, benazepril hydrochloride, captopril and fosinopril
sodium from human plasma. The log P values were 0.07,
3.3, 0.34, and 6.3, respectively. Total of 4 mL organic sol-
vents yielded recoveries of 90.60–99.38%, among which
HCT had the recoveries of 96.51–99.38%. Besides, Strata-X
33 u (RP sorbent) extracted bumetanide from urine,[62]
Celerity Deluxe SPE (RP sorbent) extracted ACE from
human plasma,[63] and strong cation exchanger mixed mode
Strata X-C[33] extracted 11 drugs including AMI, HCT and
FUR from human plasma. MeOH and water were the com-
monly used combination for conditioning or equilibrating
the cartridges.[32,62–64] Washing solution also contained
mostly the MeOH and water,[32,63] sometimes including 1%
orthophosphoric acid,[64] ammonium formate,[27] and so on.
MeOH and ACN were commonly employed for elu-
tion.[27,32,62] Owing to the lack of selectivity from traditional
commercial adsorbents, novel sorbents have been explored
for more wide and specific application.
Molecularly imprinted polymers (MIPs) have superior
selectivity and recognition characteristics based on “lock-
key” principle.[32] Yilmaz et al.[18] developed MIP-SPE for
extracting IND from human urine. IND was used as the
template molecule. The optimized conditions produced
recoveries of 80.1–80.2%. The recoveries of IND ranged
from 28.6% to 35.1% when using non-imprinted polymers
for SPE. For obtaining higher binding ability, greater iso-
merization rate constant, rapider mass constant and better
recognition capacity, a shell-structured photoresponsive hol-
low molecularly imprinted polymer (PHMIP) was designed
by Gong et al.[65] for the recognition of TRI from human
urine and serum samples (Figure 3A). The surface area,
pore volume, average pore size and binding selectivity of
PHMIP and photoresponsive surface molecularly imprinted
polymer (PSMIP) were 42.01 versus 31.52 m2/g, 0.1689 ver-
sus 0.1202 cc/g, 160.8 versus 76.26 Å and 1.56 versus
1.20 lmol/g. This novel PHMIP achieved photoregulated
uptake and release of TRI with recoveries between 93.4 and
98.5%, showing that this method provided was reliable and
practical for the extraction of diuretics.
While there is a risk of template leakage when using
MIP. Dummy-template MIP was introduced to overcome
this problem which used analogues instead of target analytes
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 9
as template molecules.[17] There was a multi-recognition
phenomenon for several compounds at the same time.
Wang et al.[36] compared three fragmental templates as
MIP-SPE for the simultaneous extraction of melamine, TRI,
trimethoprim and cyromazine which had the same fragment
2,4-diamino pyrimidinyl. 2,4-diamino-6-methyl-1,3,5-triazine
(DMT) was determined as the best dummy-template. The
greatest imprinting factor (IF) was 5.25 of DMT on DMT-
MIPs and for melamine, TRI, trimethoprim and cyromazine,
were 3.89, 2.18, 2.60, and 2.08, respectively, showing excel-
lent selectivity. Recoveries of TRI were from 86.8% to
94.8%. The process of preparing this kind of multi-recogni-
tion monolithic MIP by in-situ polymerization was time-
consuming with low yield. Precipitation polymerization had
thus been applied to design monodisperse MIP beads of
high efficiency and requiring less preparation times. Wang
et al.[17] synthesized DMT-MIPs through precipitation poly-
merization for extracting trimethoprim, cyromazine, TRI,
and melamine from urine and serum. Recoveries ranged
from 80.9% to 91.5%. This method was worth promoting
cause it not only saves time for synthesizing materials, but
also solves the problem of template leakage.
SPE adsorbents continue to progress. Magnetic materials
have been in attention for improving extraction efficiency,
reducing extraction time, and consuming less organic solvents.
Magnetic solid-phase extraction (MSPE) disperses magnetic
materials in the sample solution for adsorbing the targets, and
separates them via the external magnetic field without tedious
centrifugation or filtration[66] (Figure 2B). Magnetic nanopar-
ticles (MNPs) have size less than 100 nm. They have high
selectivity and small quantities of MNPs can achieve high
recovery of target analytes from complex samples. Metal-
organic frameworks (MOFs) as the porous materials have
high specific surface area and permanent nano-porosity.[67]
Liu et al.[19] synthesized MNPs modified Zr-based MOF
(Fe3O4@UiO-66-OH) for extracting diverse diuretics from
urine. This sorbent provided high adsorption capacity through
large surface area, hydrogen-bonding and p–p interactions
between diuretics (delocalized p-electron system) and
Fe3O4@UiO-66-OH (aromatic rings). Recoveries and EFs of
azosemide, dorzolamide, buthiazide, and brinzolamide were
93.5–103% and 93–97, respectively. Advantages of time-saving,
labor-saving and environmental-friendliness make MNPs- and
MOFs-based SPE a hot topic for researchers in recent years.
2.4.2. Micro-solid-phase extraction (l-SPE)
l-SPE is a miniaturized sample pretreatment technique.
Tens of milligrams of SPE filler are compressed into a small
column, and extraction is performed. l-SPE is an equilib-
rium-based extraction different from exhaustive extraction
mechanism of SPE.[68] m-SPE is more effective, simpler,
faster, and easier to hyphenate with chromatographic sys-
tems for automation.
The novel adsorbents have been explored considering
the disadvantages of traditional adsorbents like poor
selectivity and reproducibility, and easy tailing. Polymer
nanocomposites have polymer as matrix continuous phase
and nanofiller as dispersed phase.[20] They have easy
10 Y.-J. LIU ET AL.
Figure 3. Synthesis route for PHMIP and the corresponding PSMIP (A), preparation of Fe3O4@MIPs procedures (B) and preparation of preparation of C3N4/ZIF-
8@MIP (C).
processing, low friction and wear rate, high surface hardness
and low cost compared to pure polymeric materials. Bagheri
et al.[20] synthesized electrospun nanocomposite based on
MNPs-polybutylene terephthalate as sorbent and developed
on-line l-SPE coupled with high performance liquid chro-
matography (HPLC) for the extraction and determination of
anti-inflammatory and loop diuretic drugs (including FUR)
from urine. The recoveries for four drugs were from 78% to
91% with relative standard deviation (RSD) of 6.8–8.7 after
18 min of extraction and 120 s of desorption. Thus, this
online set-up was proved to be reproducible, fast, auto-
mated, and suitable for analysis of diuretics in real samples.
2.4.3. Dispersive solid-phase extraction (d-SPE)
d-SPE is developed based on SPE. The solid sorbent is
placed in aqueous sample solution and requires no prior
sample preparation. The extraction and desorption process
depends on shaking and centrifugation.[69] The direct con-
tact between sorbent and liquid sample avoids the condi-
tioning procedure in SPE. The extraction is time-saving,
efficient, and small particles in the matrix do not block the
cartridges. d-SPE is thus simple, widely applicable and con-
venient to operate.[32] Octadodecyl-bonded silica, graphitized
carbon black and primary-secondary amines are the univer-
sally employed commercial kits. Novel sorbents for d-SPE
with high adsorption capacity and selectivity are being syn-
thesized and discovered.[69]
Zeng et al.[38] designed MWCNTs-d-SPE for extracting
10 adulterated ingredients (including TRI, HCT, CHL and
IND) from antihypertensive functional foods (tea substitute,
tonic wine and two capsule samples). Recoveries of 81.1–
102.5% were obtained for 10 target analytes under the

optimum conditions. It reflected superior extraction com-
pared to other modes (MWCNTs-SPE and C18-d-SPE)
where recoveries for 10 drugs were less than 10.4% for the
former and recoveries for HCT and IND were 4.73% and
31.2%, respectively for the later. Arabi et al.[21] synthesized
Fe3O4@MIPs (50.0 mg) as sorbents for d-SPE for extraction
of HCT from human urine samples and got recoveries at
90.7–110.0% (Figure 3B). These novel materials exhibited
favorable extraction performance for diuretics in various
matrices and have been popularly used and further
developed.
2.4.4. Online solid-phase extraction (online SPE)
Online SPE is an automatic mode which reduces contamin-
ation, simplifies operation steps, shortens total extraction
time and directly connects with chromatography.[70] De
Wilde et al.[4] utilized turbulent flow online SPE for extract-
ing 50 diuretics and masking agents from human urine.
They compared ME under turbulent flow rate (3 mL/min)
and normal flow rate (0.3 mL/min) for loading samples
where turbulent flow rate could effectively eliminate matrix
interferences and solved the retention time shift problem.
30 mL mefruside (IS) and 300 mL urine were used. After
5 min of centrifugation, 20 mL sample supernatant was
injected to the coupled system for next analysis. Significantly
reduced extraction time, low organic solvents consumption
and extremely no ME make it promising to be widely used
in combination with chromatography.
2.4.5. Solid-phase micro membrane tip extraction
(SPMMTE)
SPMMTE can be built inhouse using micro pipette, and pip-
ette tip with sorbent fixed in polypropylene membrane. It is
cost-effective and reproducible with ease of operation.[71]
Alothman et al.[34] developed a shun shell column and
nanocomposite sorbent as SPMMTE for extracting 11 drugs
including HCT, FUR and SPI from human plasma. After
extraction and desorption of 30 min each, the recoveries
were from 85.2% to 90.4% with RSD of 0.98–1.6%. Though
acceptable recoveries were got, longer pretreatment time
showed SPMMTE still needs to be improved to be
pragmatic.
2.5. Solid-phase microextraction (SPME)
SPE provides favorable recoveries, while the amount of
organic solvents used is still large which is harmful to the
environment and operators.[64] Compared with SPE, SPME
integrates sampling, extraction, concentration, and injection
with more convenience, faster process, less organic solvents
consumption and higher efficiency. Various SPME geome-
tries have been developed to meet diverse analysis require-
ments.[72] Fiber-based SPME and thin-film SPME (TF-
SPME) are commonly used. Besides, techniques like SBME
and BAlE have also been applied for extracting diuretics.
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 11
2.5.1. Fiber-based SPME
In fiber-based SPME, the target analytes are first adsorbed
on the surface of coated fibers, and then desorbed for subse-
quent analysis and determination.[39] Desorption types
include thermal desorption (TD) and solvent desorption
(LD). It is feasible and convenient for extractions from
diverse matrices. Various coatings can be coated on fiber
cores. Targeted design can be achieved according to the
properties of analytes and matrix. Fused silica coated with
polydimethylsiloxane (PDMS), polyethylene glycol (PEG),
and divinylbenzene (DVB) are commonly used commercial
materials.[73]
However, the existing materials have problems of poor
selectivity, friability, short validity period and poor thermal
stability. The extractions of some substances cannot be
ideally met because of pertinent physical and chemical prop-
erties of analytes. Therefore, novel coating fibers have always
been developed. Wang et al.[39] synthesized disposable poly-
styrene/poly(ethylene glycol) diacrylate (PS/PEGDA) coating
for quartz fibers to extract pharmaceuticals from water
matrix. Synthesized fibers exhibited excellent chemical and
mechanical stability through dense, uniform, and porous
surface. Recoveries of HCT and FUR were 95.4% and
100.2%, respectively. EFs were 409 for HCT and 878 for
FUR. Extraction efficiency of PS/PEGDA fibers for FUR was
278 times higher than PDMS fibers. PDMS fiber showed no
detectable extraction for HCT. Comparative experiments
showed that the new material had superior extraction and
enrichment properties. Nevertheless, few novel SPME fibers
have been reported for the extraction of diuretics in recent
years, which suggested that the synthesis of novel fibers is a
potential research direction.
2.5.2. TF-SPME
Limitations of slow mass transfer and low extraction effi-
ciency of fiber SPME are revealed when sample volume is
large. TF-SPME is introduced to overcome these disadvan-
tages. It disperses the extractant onto flat film sheet without
increasing the coating thickness (compared with fiber geom-
etry). The large specific surface area improves analytical sen-
sitivity and shortens extraction time.[72] Commonly used
phases are C18 and PDMS. They however fail to satisfy the
requirements of all prohibited substances regulated by
WADA. Besides, PDMS can swell upon introduction of
some liquid chromatographic solvents.[68] New phases
should thus be applied for TF-SPME. Sobczak et al.[74] com-
pared 12 HPLC-compatible coatings for extracting 48 com-
monly used prohibited compounds (including FUR, HCT,
CHL, CTZ, and CAN) in sports. C8 þ CN (1:1)-type coating
was the best for extracting 48 substances with RSD of 1.5–
4.8%. C8 þ CN (1:1)-type coating provided hydrophobic
interactions like C18 in addition to dipole–dipole and p–p
interactions to increase the analyte coverage and extraction
efficiency.
Biocompatible SPME coatings prepared by immobilizing
sorbents with polyacrylonitrile (PAN) can extract drugs
from complex biological matrices. Automatic systems have
been introduced to achieve high throughputs and thus save
12 Y.-J. LIU ET AL.
time. Reyes-Garces et al.[75] selected HLB Oasis particles-
PAN as the coating of TF-SPME for extracting 25 doping
agents including FUR (log P from 2 to 6.8) from human
plasma. The preparation time of each sample was 1.5 min
when combined with 96 thin film processing of automatic
system. Absolute MEs were obtained from 100% to 120%
for 24 out of 25 substances. This method was proved to be a
favorable choice for high throughput treatment of multiple
drugs in complex biological matrices with minimum sample
handling (1.08 mL).
2.5.3. SBME
SBME as a new extraction technique adopts a section of nar-
row hollow fiber to fill in the sorbent material. Both ends of
fiber are sealed and placed in sample solution. The analytes
diffuse between the fiber and sample solution owing to
membrane porosity, and extracted by the sorbent under vig-
orous stirring. The desorption of target analytes can be
achieved in an appropriate organic solvent.[76] Compared
with SPME, this method has easier operation, requires no
desorption unit, less expensive, simple and environment
friendly. AL-Hashimi et al.[23] employed SBME for extract-
ing diuretics (FUR and SPI) from human urine without any
further cleaning procedure. Only 2 mg C18 sorbent and
250 lL MeOH (elution) were consumed. While recoveries of
only 14.6–37.4% were got for FUR and SPI, which suggested
that this method was unsuitable for extracting polar
compounds.
Another similar extraction technique, BAlE, is more suit-
able for extracting medium-polar to polar substances than
SBME.[77] Figure 2C illustrates the procedure of BAlE. It
uses bar-shaped polypropylene apparatus coated with
adsorbent phase, selected according to the physicochemical
properties of target analytes.[78] Pretreatment process
includes two procedures: microextraction and back-extrac-
tion performed through LD. This extraction setup is easy to
prepare, economical, consumes trace organic solvents and
can apply different sorbents.[78] Almeida et al.[22] used
BAlE coupled with micro-liquid desorption (100 lL) for
extracting six pharmaceuticals including FUR from urine.
The complex and time-consuming solvent conversion pro-
cess was eliminated and back-extraction stage could be com-
pleted in one step. Polymer P5 (0.9 mg) was selected as the
sorbent to attain high selectivity and efficiency. The average
recovery was 91.4% (FUR) and 101.0% (ketoprofen) which
suggested that BAlE could be an alternative technique for
effectively extracting diuretics.
2.6. QuEChERS
QuEChERS refers to quick, easy, cheap, effective, rugged,
and safe method, first proposed by Anastassiades et al.[79] in
2003. It was initially used for analyzing pesticide residues in
fruits and vegetables. The steps can be summarized as fol-
lows: (1) crush samples; (2) use ACN for extraction and sep-
aration; (3) add salt (e.g. MgSO4) to remove water; and (4)
add sorbent (e.g. ethylenediamine-N-propylsilane (PSA)) to
remove impurities. The method is cheap, no requirement of
special devices, high sample throughput, low organic solvent
consumption and ease of operation.[60] Chen et al.[40] used
QuEChERS for extracting diuretics from animal-derived
foods. After optimizations, ACN (15 mL), Na2SO4 (2 g), and
sorbents mixture of MgSO4 (600 mg), PSA (100 mg) and C18
(100 mg), recoveries for HCT, SPI, CAN, CTZ, and ACE
were from 72.1% to 118.1%.
2.7. Summary of pretreatment methods
The sample pretreatment methods need comprehensive ana-
lysis regarding types of samples being tested physical and
chemical characteristics of analytes, and matrix effects to
select the best pretreatments for achieving fast, efficient and
environment friendly analysis. PPT is the simplest method
for processing biological samples with low cost and easy
implementation. The selectivity of PPT is poor and co-pre-
cipitation of endogenous substances leads to serious ME
which limits its applications. Compared with PPT, LLE has
better selectivity and wider applicability in sample pretreat-
ments. It however consumes large quantities of organic sol-
vents which are harmful to environment and the operators,
and increase the cost. In contrast, LPME is faster and envir-
onment-friendly. Various modes such as HF-LPME and
DLLME have advantages such as suitable for extractions
from dirty complex samples and rapid analysis respectively.
SPE is extensively used for extracting diuretics from physio-
logical samples. It consumes less organic solvents than LLE
but fails in favorable inter-batch repeatability as it is difficult
to ensure exactly the same properties of sorbents. The inno-
vations in SPE sorbents such as MNPs and MWCNTs have
always been focused. SPME retains the advantages of SPE
and does not require column filler. Moreover, the extraction
process needs no organic solvents, thus making it environ-
ment friendly. The process is rapid and its various forms
such as fiber-based SPME, TF-SPME and SBSE can be
applied to analytes of diverse physicochemical properties
present in various matrices and obtain higher recovery. The
pros and cons of diverse extraction methods were concluded
in Table S2.
3. Analytical methods
The sub-standard drugs are common in circulation market,
and inaccurate dosage of drug components may pose danger
or irreversible conditions to human body.[80] Besides, for the
doping detections in athletes, the content of diuretics in bio-
logical samples is small and some diuretics may degrade
during placement. Therefore, the detection strategies and
instruments ought to be sensitive, rapid, and reliable.
Common detection techniques include liquid chromatog-
raphy–mass spectrometry (LC–MS) or tandem mass spec-
trometry (MS/MS), LC or capillary electrophoresis (CE)
coupled with detectors such as ultraviolet detector (UVD),
fluorescence detector (FLD) and refractive index detector
(RID), thin layer chromatography (TLC), sensors, and others
(Figure 4). The following sections summarize determination

Figure 4. Analytical methods for diuretics in diverse matrices.
methods of diuretics in diverse matrices such as drug dosage
forms, urine, and blood specimens. The advantages and dis-
advantages of employed methods are discussed (Table S3).
3.1. LC Methods for diuretics
Browsing the research on determining diuretic components
since 2015, LC proves the frequently used separation meth-
odology.[38,43,81] LC based methods are suitable for stable or
unstable compounds of high boiling points and high
molecular weights which account for 80% of the organic
matter. Some reports on determination of diuretics are sum-
marized in Table 2.
3.1.1. Commonly used LC columns
LC separation modes can be divided into normal-phase
whose stationary phase is polar and RP whose stationary
phase is non-polar. Many chromatographic columns are
used for separating diuretics owing to their diverse physico-
chemical characteristics. C18 columns are the most prevailing
in RPLC. They possess universality and large retention value
for separating and analyzing numerous organic compounds
such as non-polar, weak, and medium polar small molecules,
as well as polypeptides and proteins. Studies had reported
C18 columns are the most widely used for separating diu-
retics such as SPI, HCT and FUR.[23,97,98] Besides, C8 col-
umns[89] whose retention capacity is smaller than C18
columns, cyano columns[91,93] and phenyl-hexyl
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 13
columns[41,99] were also used in separating diuretics. Sugar
LC columns[82] were used to separate mannitol and sorbitol.
In recent decades, the LC systems have been developed
and fine-tuned to convert HPLC to ultrahigh-performance
liquid chromatography (UPLC). The conversion essence is
reflected in reduction of filler particle sizes, that is, from 3
to 5 lm to <2.5 lm.[106] Though HPLC is less expensive
and more convenient, UPLC improves analysis efficiency
and shortens the separation time.[43] Mahrouse et al.[61]
employed UPLC with SB-C18 column (50  2.1 mm, 1.8 mm)
for separating benazepril hydrochloride, fosinopril sodium,
captopril and HCT from plasma where injection volume was
7.5 mL and runtime of only 1.5 min. Compared with HPLC
using C18 Zorbox eclipse plus (150  4.6 mm, 5 mm)[47] for
separation of amlodipine, olmesartan and HCT, whose total
runtime was 4.2 min, UPLC achieved better and rapid
separation.
In addition to the column, mobile phase plays a role in
chromatographic separations. The mobile phase composition
is usually fixed like MeOH-water,[8,22,101] ACN-water[27,32,63]
or MeOH-ACN.[13,41,47] Ethanol has also been employed as
mobile phase in the perspective of Green Analytical
Chemistry.[84,107] Additives such as acid, alkali and salt are
added to adjust pH for improving the peak shape and elimi-
nating the tailing. In separation procedures of diuretics,
commonly used additives are ammonium formate,[7,41,47]
formic acid,[32,63,101] orthophosphoric acid,[22,86–88] potas-
sium dihydrogen phosphate,[83,84,100] heptafluorobutyric
acid,[108] and triethylamine.[109] Among them, phosphate is
widely used buffer in LC–UV methods, while pH cannot
Table 2. HPLC based method for diuretics.
14

Retention
Sample matrix Analytes Analysis method Column Mobile phase Analysis condition time (min) Linear range LOD LOQ Ref.
[43]
Dietary FUR, HCT, CHL, AMI, ACE UPLC-QqQ-MS/MS UHPLC Zorbax 0.05% formic acid in ESI, ±, MRM, SRM 0.36–14.50 3.90–2000 ng/mL 0.09–1.21 ng/mL 0.31–3.92 ng/mL
Supplements and SPI model SB-C18 water/ACN;
column gradient elution;
(50  2.1 mm, 0.6 mL/min
1.8 lm)
Y.-J. LIU ET AL.

[38]
Dietary CHL, TRI, HCT and IND HPLC-VWD Phenomenexw 0.03 mol/L KH2PO4 220 nm 4.45–14.0 0.1–100 mg/mL 14–53 ng/mL 48-178 ng/mL
Supplements Luna C18 solutions (pH 3.0) (A)
column and ACN (B);
(250  4.6 mm, gradient elution;
5 lm) 0.80 mL/min
[82]
Low-calorie dessert Sorbitol HPLC-RID ShodexVR Sugar Distilled water; Column 47.14 0.1–5 mg/mL 0.17 mg/mL 0.56 mg/mL
foods SP0810 (300 mm isocratic elution; temperature:
 8.0 mm i.d.) 0.5 mL/min 80  C
column RID
2þ
with Pb temperature:
35  C
[81]
Tablets CHL HPLC-UVD Phenomenox, Water: ACN (55:45, v/v), 250 nm 3.80 0.5–12.5 mg/mL 15 ng/ml 80 ng/ml
Gemini C18 pH 3.4, adjusted with
column ortho phosphoric acid;
(250  4.6 mm, isocratic elution;
5 mm) 1 ml/min
[83]
Tablets IND HPLC-UVD BDS Hypersil C18 0.05 M potassium 215 nm 6.097 0.5–20 mg/mL 110 ng/mL 340 ng/mL
column dihydrogen phosphate
(100  3 mm, buffer (pH 2.6)-MeOH
5 lm) (50:50, v/v);
isocratic elution;
0.6 mL/min
[84]
Tablets HCT and AMI HPLC-UVD ODS-C18 0.05 M KH2PO4: ACN: 260 nm 1.24–1.25 1–50 mg/mL 240–280 ng/mL 720–850 ng/mL
column triethylamine (90: 10:
(250  4.6 mm, 0.3 v/v/v, pH 3.6);
5 lm) isocratic elution;
1.5 mL/min
[85]
Tablets TOL HPLC-UVD Sunsil C18 column ACN: water (45:55, v/v); 266 nm 4.7 35–175 mg/mL 310.2 ng/mL 940 ng/mL
(150  4.6 mm, isocratic elution;
5 lm) 1.0 ml/min
[86]
Tablets and TOR, FUR, azosemide, HPLC-UVD Shimadzu HC-C18 ACN with 0.05 mol/L 278 nm 4.8–18.5 2.5–150 mg/mL 50–200 ng/ml n.d.
injections etacrynic acid and column KH2PO4 (pH 4.0,
bumetanide (150  4.6 mm, adjusted with diluted
5 mm) phosphoric acid) (36:
64, v/v);
isocratic elution;
1.0 mL/min
[87]
Tablets Xipamide, TRI and HCT HPLC-VWD Agilent Hypersil 0.03 mol/L 220 nm 0.961–1.853 0.195–100 mg/mL 12–24 ng/mL 40–80 ng/mL
BDS C18 column orthophosphoric acid
(150  4.6 mm, (pH 2.3) and ACN
5 lm) (ACN) (1:1, v/v);
isocratic elution;
2.0 mL/min
[88]
Tablets FUR and SPI HPLC-DAD Kinetex C-18 MeOH-water (75:25, v/v, 240 nm 2.58–3.88 0.30–12 mg/mL 80–170 ng/mL 300–450 ng/mL
column pH 3.0, adjusted with
(100  4.6 mm, phosphoric acid);
2.6 mm) isocratic elution;
0.4 mL min
[89]
Tablets ACE HPLC-DAD C8 column 10mM potassium 265 nm 6.8 20–120 mg/mL 37.60 ng/mL 113.96 ng/mL
(250  4.6 mm, dihydrogen phosphate
5 mm) buffer (pH 3, adjusted
 with O-phosphoric
acid), ACN and water
(30:20:50);
isocratic elution;
0.8 mL/min
[90]
Tablets HCT, FUR, TOR, SPI HPLC-DAD ACE C18 100 Å 0.05 M phosphate buffer 230 nm < 10 15.5–292 mg/mL 3.7–16.6 lg/mL 11.2–50.4 lg/mL
and CAN column (pH 3.0)-ACN-MeOH
(250  4.6 mm, (30:20:50, v/v/v);
5 lm) isocratic elution;
1.0 mL/min
[91]
Capsules ACE HPLC-DAD Agilent Zorbax SB- MeOH: water: phosphoric 254 nm 4.601 0.5–82 mg/mL n.d. 497.58 ng/mL
CN column acid; 1:9:0.1 v/v/v (A)
(150  4.6 mm, and MeOH: water:
3.5 mm) phosphoric acid; 4:6:0.1
v/v/v (B);
gradient elution;
1.0 mL/min
[92]
Tablets HCT HPLC-PDA Thermosil C18 20 mM potassium 248 nm 1.823 6.25–100 mg/mL 4 ng/mL 13 ng/mL
column dihydrogen
(100  4.6 mm, orthophosphate buffer:
3.7 lm) MeOH (40:60, v/v);
isocratic elution;
1.0 ml/min
[93]
Tablets HCT MELC-UVD Shim-pack cyano 0.2 M sodium dodecyl 210 nm 3.6 0.05–5 mg/mL 20 ng/ml 50 ng/ml
column sulfate, 1% octanol,
(150  4.6 mm, 10% n-propanol and
5 mm) 0.3% triethylamine in
0.02 M phosphoric
acid, pH 3.5;
isocratic elution;
1 mL/min
[94]
Human urine MAN HPLC-QqQ-MS/MS Eclipse XDB-C18 Initial test: 10 mM ESI, þ, MRM < 18 0.5–1000 mg/mL 5 ng/mL (initial 15 ng/mL (initial
column aqueous ammonium test) test)
(100  2.1 mm, formate buffer which 50 ng/mL 50 ng/mL
3.5 lm) (initial was adjusted to pH 3.5 (confirmation) (confirmation)
test) with formic acid (A)
Poroshell 120 and ACN (B)
PFP column Confirmation: 10 mM
(150  2.1 mm, aqueous ammonium
2.7 lm) formate buffer which
(confirmation) was adjusted to pH 3.5
with formic acid (A)
and MeOH (B);
gradient elution;
0.4 mL/min
[41]
Human urine TOR, HCT, TRI, TOR-M HPLC-Q-Orbitrap- TF Accucore 2 mM aqueous ESI, ±, FS 13.5 n.d. 0.001–0.1 mg/L 0.01–1 mg/L
(HOOC-) and CAN MS/MS PhenylHexyl ammonium formate
column plus 0.1% formic acid,
(100  2.1 mm, pH 3 (A) and 2 mM
2.6 mm) aqueous ammonium
formate with ACN:
MeOH (50:50, v/v; 1%
water) plus 0.1%
formic acid (B);
gradient elution;
0.5 mL/min (0–10 min),
0.8 mL/min (10–
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY

13.5 min)
[95]
Human urine HCT HPLC-DAD 226 nm <4 25.3–78.2 mg/mL 0.52 mg/kg n.d.
(continued)
15
Table 2. Continued.
16

Retention
Sample matrix Analytes Analysis method Column Mobile phase Analysis condition time (min) Linear range LOD LOQ Ref.
Prontosil 8.50 g orthophosphoric
Spheribond acid 85% diluted to
ODS-1 Bischoff 1000 mL with water
column and 280 mL
(125  4.0 mm, hexylamine (A)
Y.-J. LIU ET AL.

3 lm) 4.25 g orthophosphoric

acid 85% dissolved in
45.75 g of water, 351 g
of ACN and 140 mL
hexylamine (B);
gradient elution;
1 mL/min
[23]
Human urine FUR and SPI HPLC-DAD Inertsil ODS-3 C18 20% ACN (A) and 80% 230 nm 3.73–4.97 10–100 ng/mL 1.45–2.09 ng/mL 4.85–6.92 ng/mL
column ACN (B), containing
(150  4.6 mm, 0.25% acetic acid for
5 mm) both solvents;
gradient elution;
1.5 mL/min
[22]
Human urine FUR HPLC-DAD Mediterranea Sea- Mixture of water with 230 nm 7.6 0.1–24 mg/mL 6.0 ng/mL 20.0 ng/mL
18 column 0.1% phosphoric acid
(150  2.1 mm, (A) and MeOH (B);
5 lm) gradient elution;
0.2 mL/min
[96]
Human urine SPI and CAN UPLC-PDA Acquity UPLC HSS KH2PO4 10 mM and 237.9 nm for SPI 8.926–9.028 180–1529 mg/mL 78.12 ng/mL 156.25 ng/mL
T3 column 0.025% phosphoric 284.5 nm
(150  2.1 mm, acid (A) and ACN (B); for CAN
1.8 lm) gradient elution;
0.4 mL/min
[20]
Human urine FUR HPLC-UVD MZ analytical ACN and filtrated double- 238 nm 4.6 1–400 mg/mL 1.6 ng/mL 4.0 ng/mL
column ODS-3 distilled water (1:1,
(250  4.6 mm, v/v) with 0.1% H3PO4;
5 mm) isocratic elution;
1.0 mL/min
[97]
Hair HCT and UPLC-QqQ-MS/MS Acquity UPLC HSS 1.0 mM ammonium ESI, , MRM 3.87–4.08 5–2000 pg/mg 3 pg/mg 5 pg/mg
hydroflumethiazide C18 column acetate in water (A)
(150  2.1 mm, and 1.0 mM
1.8 mm) ammonium acetate in
MeOH (B);
gradient elution;
0.3 mL/min
[98]
Ocular tissues SPI and UPLC-QqQ-MS/MS Waters XBridge 0.1% formic acid in water: ESI, þ, SIR 2.35–2.93 5–1000 lg/mL n.d. 5 ng/mL
CAN BEH C18 column MeOH (48:52, v/v);
(50  2.1 mm, isocratic elution;
2.5 mm) 0.45 mL/min
[99]
Oral fluid CAN, SPI, FUR, HCT, TOR, HPLC-TF Q- TF Accucore 2mM aqueous ammonium ESI, ±, FS and data < 13.5 n.d. 0.5–50 ng/mL 5–100 ng/mL
TRI and xipamide Orbitrap-MS/MS PhenylHexyl formate containing dependent
column formic acid (0.1%, v/v, acquisition
(100  2.1 mm, pH 3) (A) and 2 mM (DDA)
2.6 mm) aqueous ammonium
formate with
ACN:MeOH (50:50, v/v)
plus 0.1% formic acid,
and water (1%, v/v)
(B);
gradient elution;
0.5 mL/min (0–10 min),
 0.8 mL/min (10–
13.5 min)
[100]
Infusions and FUR HPLC-DAD SinoChrom ODS-BP 50mM KH2PO4 buffer (A) 280 nm 8.76 6–240 mg/mL 240 ng/mL 900 ng/mL
injections column and ACN (B)
(150  4.6 mm, gradient elution;
5.0 lm) 1.0 mL/min
[8]
Human plasma TOR HPLC-Q-MS/MS Gl Sciences Inertsil MeOH with 10 mM ESI,  3.68 1–2500 ng/mL 0.5 ng/mL 1 ng/mL
ODS-3 column ammonium formate
(100  2.1 mm, (60:40, v/ v);
5.0 mm) isocratic elution;
0.2 mL/min
[27]
Human plasma HCT HPLC-QqQ-MS/MS Hypersil Gold C18 ACN-5.0 mM ammonium ESI, , MRM, 0.83 0.50–250.0 ng/mL n.d. 0.50 ng/mL
column formate, pH 4.5 (85:15,
(50  3.0 mm, v/v);
5 mm) isocratic elution;
0.550 mL/min
[32]
Human plasma TOL and its metabolites HPLC-QqQ-MS/MS Waters nova-pak ACN, water and formic ESI, þ, MRM 2.5 1–500 ng/mL n.d. 1 ng/mL
C18 column acid (65:35:0.25, v/v/v);
(150 isocratic elution;
3.9 mm, 5 lm) 0.8 mL/min
[63]
Human plasma ACE HPLC-QqQ-MS/MS Hypurity advance 0.1% formic acid buffer ESI, ±, MRM 0.9 50.3–12046 ng/mL n.d. 50.3 ng/mL
column and ACN (30:70, v/v);
(50  4.6 mm, isocratic elution;
5 mm) 0.80 mL/min
[7]
Human plasma TOL and its major HPLC-QqQ-MS/MS Octadecylsilyl 2.5 mmol/L ammonium ESI, þ < 15 3.125–1000 ng/mL n.d. 0.125–31.25 ng/ mL
metabolites column acetate (A) and ACN
(250  2.0 mm, (B);
3 mm) gradient elution;
0.3 mL/min
[101]
Human plasma SPI HPLC-QqQ-MS/MS Cadenza CD-C18 0.1 % formic acid in ESI, þ, MRM 4.05 0.5–150 ng/mL n.d. 0.5 ng/mL
column water: MeOH (30:70,
(100  3.0 mm, v/v);
3 mm) isocratic elution;
320 mL/min (0–3.2 min),
320–180 mL/min (3.2–
3.5 min), 180 mL/min
(3.5–6.7 min); 180–
320 mL/min (6.7–
7.0 min)
[47]
Human plasma HCT HPLC-QqQ-MS/MS Zorbax SB-Aq 10 mM ammonium ESI, , MRM, SRM 4.2 2–150 ng/mL n.d. 0.10–5.00 ng/mL
column formate containing
(150  4.6 mm, 0.1% formic acid:
5 mm) MeOH: ACN (35:50:15,
v/v/v);
isocratic elution;
0.7 mL/min
[102]
Human plasma Conivaptan HPLC-QqQ-MS/MS RP C18 column ACN and 10 mM ESI, þ, MRM 4.0 5–500 ng/mL 1.52 ng/ml 5 ng/ml
(50  2.1 mm, ammonium formate
1.8 lm) (40:60 v/v, pH 4.0);
isocratic elution;
0.2 mL/min
[103]
Human plasma HCT and CAN UPLC-QqQ-MS/MS Hypersil Gold 100% ACN (A) ESI, ±, SRM 2.5 5–500 ng/mL 0.02–2.82 ng/mL 0.05–8.54 ng/mL
column C18 0.1% formic acid in
(50  2.1 mm, ultrapure water (B);
1.9 mm) isocratic elution;
0.27 mL/min
[6]
Human plasma and IND UPLC-QqQ-MS/MS Thermo BDS Mixture of water and ESI, þ, MRM 2.39–2.41 1–250 ng/mL n.d. 1.00 ng/mL
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY

whole blood Hypersil C18 MeOH (90:10, v/v)

column containing 0.05%
(continued)
17
Table 2. Continued.
18

Retention
Sample matrix Analytes Analysis method Column Mobile phase Analysis condition time (min) Linear range LOD LOQ Ref.
(100  4.6 mm, ammonium acetate
2.4 mm) and 0.2% formic acid
(A) and MeOH
containing 0.05%
ammonium acetate
Y.-J. LIU ET AL.

and 0.2% formic acid

(B);
gradient elution;
0.65 mL/min
[61]
Human plasma HCT UPLC-QqQ-MS/MS Agilent SB-C18 MeOH: water containing ESI, , MRM 0.4 5–400 ng/ml n.d. 5 ng/ml
column (50 mm 0.1 % formic acid in
 2.1 mm, water (95: 5, v/v);
1.8 mm) isocratic elution;
0.3 ml/min
[104]
Human plasma HCT UPLC-QqQ-MS/MS XBridge BEH C18 0.1% formic acid in ESI, , MRM 1.20 5–400 ng/ml n.d. 5.0 ng/mL
column ammonium acetate
(50  2.1 mm buffer (0.02 M, pH 3.5)
ID, 5 lm) (A) and MeOH (25:75,
v/v) (B);
isocratic elution;
0.6 mL/min
[13]
Human plasma CHL HPLC-QTRAP QqQ- Thermo Syncronis- 10 mM ammonium ESI, , MRM < 2.5 1–4000 ng/ml n.d. 1 ng/mL
MS/MS C18 column acetate, pH 4, adjusted
(100  4.6 mm, using acetic acid (A)
5 mm) and
mixture of MeOH-ACN,
50:50, v/v (B);
isocratic elution;
0.7 mL/min
[84]
Human plasma and FUR, SPI and CAN HPLC-UVD ZOBRAX Eclipse Ethanol: deionized water 254 nm 1.64–4.42 2–60 mg/mL 0.485–1.333 mg/mL 1.6–4.0 mg/mL
tablets Plus C18 column (45: 55, v/v) pH 3.5,
(100  4.6 mm, adjusted with glacial
3.5 lm) acetic acid;
isocratic elution;
1 ml/min
[105]
Human plasma and HCT HPLC-PDA Phenomenex RP- 10 mM phosphoric acid- 271 nm 9.05–9.13 0.0125–2.5 mg/mL 2–4 ng/mL 6–12 ng/mL
urine C18 column 0.1% triethylamine (A)
(250  4.6 mm, and ACN (B);
5 lm) gradient elution;
1 mL/min
[33]
Human plasma and HCT, AMI and FUR UPLC-PDA-FD BEH C18 column pH 4.5 buffer solution (A) 271 nm (HCT) <6 0.08–5 mg/L n.d. 0.30, 0.08,
tablets (50  2.1 mm, and MeOH (B); kex: 363 (AMI), 0.30 mg/mL
1.7 lm) gradient elution; 235 (FUR)
0.4 mL/min kem: 415 (AMI),
398 (FUR)
n.d., not found.

exceed 7, otherwise, phosphate speeds up the dissolution of
silica and reduces the life cycle of silica-based HPLC col-
umns. For LC–MS, nonvolatile buffer salts like phosphate
are prohibited as they block the spray needle and affect sub-
sequent analysis.[110] HPLC has superior separation capacity,
while it consumes large organic solvents and fails to charac-
terize two substances with the same retention time accur-
ately. Accordingly, to realize greener and improve qualitative
capabilities, novel forms of HPLC and HPLC coupled with
different detectors are proposed and prevailing.
3.1.2. Development of HPLC
Micellar liquid chromatography (MLC) has been developed
to reduce the organic solvents consumption and to meet the
concept of green chemical strategy. MLC is the developed
form of HPLC whose mobile phase is an aqueous surfactant
solution with concentrations higher than critical micelle
concentration and is a heterogeneous dispersion system.[111]
MLC system has three interfaces composed of stationary
phase-mobile phase-micelle-stationary phase, and three par-
tition coefficients affecting the separation process. Other
advantages include high reproducibility, selectivity, low cost,
environment friendliness, rapid gradient elution and no pre-
treatments for biological samples.[112] Tolba et al.[111]
applied MLC-time-programmed FLD for determining xipa-
mide (XIP) and its degradation product 2,6-xylidine from
human urine. Mobile phase contained 0.1 M sodium dodecyl
sulfate (SDS) and 15% n-propanol (pH 5.0) which was
greener than 65% MeOH used in conventional HPLC. Urine
samples could be injected directly without pretreatment. The
injection volume was 20 lL. LOD and limit of quantification
(LOQ) of XIP were 0.13 and 0.39 lg/mL, and those of 2,6-
xylidine were 0.03 and 0.10 lg/mL, respectively. Satisfactory
results exhibited that this environment-friendly method pro-
posed could be an alternative for detecting diuretics in bio-
logical samples.
Microemulsion liquid chromatography (MELC) is
another emerging technique as a type of HPLC with microe-
mulsion as mobile phase. Microemulsion mobile phases are
composed of water phase, oil phase, surfactant, and co-sur-
factant. They can be transparent or translucent, low viscous,
isotropic and thermally stable water oil mixed system.
Microemulsions can dissolve substances from hydrophilic to
hydrophobic.[113] Compared with conventional HPLC,
MELC has advantages such as (i) microemulsions reduce
consumption of organic solvents and thus greener and eco-
friendly, (ii) no pretreatments of biological samples required
as microemulsions can dissolve proteins, and (iii) analyze
analytes needing detection at low UV wavelength but with-
out strong chromophores.[114] Li et al.[115] applied MELC-
UV for determining HCT and losartan potassium in
Osmotic Pump Tablets. The optimum mobile phase was
composed of 95% (v/v) of 3.0% (w/w) SDS, 6.0% (w/w) n-
butanol, 0.8% (w/w) n-octane, 90.2% (w/w) water, and 5%
(v/v) ACN (pH 5.0). Only 67.8 mL mobile phase was con-
sumed in this proposed method contrary to HPLC where
organic solvent consumption was 350 mL for simultaneously
analyzing HCT and losartan potassium,[116] showing MELC
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 19
method being more environment friendly. LOD and LOQ of
HCT were 30 and 100 ng/mL, respectively and RSD less
than 1.0%. MELC is thus a favorable alternative method for
routine drugs analysis.
3.1.3. MS detectors
MS is an excellent analytical tool at present because of its
high sensitivity, accuracy, throughput and selectivity. MS
consists of ionization source, analyzer, and detector.
Ionization of sample molecules is necessary for MS deter-
mination. Reviewing the literatures since 2015, it is found
that electrospray ionization (ESI) is the most commonly
used[41,94,98] which accounts for more than 90% of quantita-
tive analysis of diuretic components studied by MS. ESI as
ionization source has following advantages: (i) provide
accurate molecular weights and structural information at the
same time, (ii) fast, (iii) two ionization modes, that is, posi-
tive ionization (PI, þ) mode for alkaline analytes and nega-
tive ionization (NI, -) mode for acidic analytes, (iv)
effectively combined with chromatographic systems, and (v)
allowing the use of analyzers such as quadrupole.[117]
Mass analyzer is critical to MS which defines the sensitiv-
ity and resolution of MS.[118] At present, there exists low-
resolution mass spectrometry (LRMS) and high-resolution
mass spectrometry (HRMS). Difference being the molecular
mass provided by the former as a mass number accurate to
2 decimal places while that of latter is 4-6 decimal places.
HRMS can thus distinguish compounds with same nominal
mass.[97,99] For LRMS, the resolution power is <10,000
FWHM and mass accuracy >5 ppm, while with HRMS, it is
>10,000 FWHM and <5 ppm, respectively.[119] LRMS
employs quadrupole (Q) and triple quadrupole (QqQ) ana-
lyzers for routine laboratory analysis, while HRMS uses time
of flight (TOF) and Orbitrap, together with hybrids like Q-
TOF and Q-Orbitrap, universally used in research laborato-
ries.[8,99,102] Characteristics of different mass spectrometers
were overviewed in Table S4.
Q is the simplest and quality analyzer at present. It is
cheap, easy to maintain, and has good quantitative ability. It
has insufficient qualitative ability, low resolution, and fails
to perform MS/MS analysis.[118] For improving the qualita-
tive capacity, MS/MS analysis can be achieved by attaching
Q to other mass analyzers such as additional Q (QqQ), TOF
(Q-TOF) and Orbitrap (Q-Orbitrap). QqQ mass analyzer is
the most prevailing and accounts for more than 90% of the
detections of diuretics from diverse samples by LC-
MS.[7,27,32] It has multiple scanning modes such as precursor
ion scanning, neutral loss scanning, product ion scanning,
selective reaction monitoring (SRM) and multiple reaction
monitoring (MRM), which are significant for studying the
structures of characteristic groups. MRM with strong specifi-
city, sensitivity, wide linear dynamic range, accuracy, and
reproducibility, is frequently used in diuretics detection by
LC-MS. It exhibits superiority when simultaneously deter-
mining various compounds as analytes can be identified not
only by precursor ions but also by product ions.[43,104] MS
detectors have superior sensitivity that Lo Faro et al.[120]
applied HPLC-QqQ-MS/MS with MRM mode to detect
20 Y.-J. LIU ET AL.
ACE in urine samples and achieved LOD of 1.3 ng/mL. This
is a sensitivity that other detectors (usually mg/mL) cannot
achieve.
HRMS with high resolution and specificity has developed
new functions such as broad screening analysis, unknown
analysis, and detection of transformation products of pollu-
tants and other chemicals.[60] Ki et al.[121] developed LC-Q-
TOF-MS coupled with neutral loss scan for screening sulfo-
namides diuretics in various supplements. LODs of diuretics
including CTZ, ACE, HCT, FUR, CHL, tolazamide and
TOR ranged from 0.04 to 10.77 ng/g. Orbitrap mass analyzer
is a relatively new mass analyzer appeared first in 2005. It
has overcome disadvantages like low sensitivity and reso-
lution of TOF. Orbitrap mass analyzer is combined with ion
implantation process and is the first high-performance mass
analyzer to capture ions in electrostatic field, achieving high
mass accuracy, sensitivity and resolution.[122] It however
cannot be used alone for MS/MS analysis and commonly Q
is attached with Orbitrap. Abushareeda et al.[123] used Q-
Orbitrap MS to screen known or unknown prohibited sub-
stances and their metabolites in human urine for doping
control analysis. After urine pretreatment, Q-Orbitrap MS in
full scan (FS) with positive/negative switching mode was
employed for detecting urine drugs. LODs of ACE, AMI,
azosemide, bendroflumethiazide, benzthiazide, bumetanide,
CAN, chlorothiazide (CTZ), CHL, conivaptan, FUR, HCT,
IND, mozavaptan, TOL, TOR, xipamide, and TRI were less
than 2 ng/mL. The ability to simultaneously perform tar-
geted and non-targeted screening makes the application of
HRMS more widespread and popular.
While ME is the notable failing in LC-ESI-MS which can
bring signal enhancement or suppression due to co-eluting
molecules, resulting in quantitative inaccuracy and increase
of LOD.[124] Some methods have been proposed to reduce
or eliminate ME influence. The common solution is to use
isotopic internal standard (IS).[7,13] Ramakrishna et al.[13]
used HPLC-MS/MS to determine CHL in human plasma
using HCT as IS. IS normalized matrix factor (measure ME,
the closer it is to 1, smaller the ME is) was 1.02 and 0.98 at
two concentration levels, demonstrating the matrix has little
influence. Besides, ME can be solved by optimizing the sam-
ple pretreatment process.[35,36,39] Narapusetti et al.[63] used
SPE-HPLC-MS/MS for analyzing ACE in plasma samples.
Orochem celerity deluxe SPE cartridge was optimum for
this experiment and ACE d3 was selected as IS. The com-
bined usage of two methods minimized or even eliminated
the ME and improved precision (1.56%) and accuracy
(97.6%). ME can also be solved by adjusting the liquid phase
separation conditions, reducing injection volume or diluting
the sample, and selecting other ion sources.[124]
3.1.4. LC – other detectors
MS is the first choice for analyzing complex drug mixtures
because of its high sensitivity and identification ability.
UVD, FLD and RID are also employed for detecting diu-
retics with characteristics like photometric and fluorescent.
These detectors are less expensive and easier to operate than
MS. UVD is the common HPLC detector due to majority of
organic and inorganic substances showing absorptions in
UV (or visible) region at certain wavelength or wavelength
range. UVD has high sensitivity, wide linear range and low
noise and is not sensitive to ambient temperature, mobile
phase composition changes and flow rate fluctuations.[110] It
can thus be used for isocratic and gradient elution. There
are three UVD types: fixed wavelength detector (254 nm),
variable wavelength detector (VWD), and photodiode array
detector (PDA or DAD). The latter two are prevalent which
ensure high sensitivity in wide wavelength range. ABD EL-
HAY et al.[87] used HPLC-VWD for determining TRI and
HCT in their bulk powders and commercial tablets at
220 nm. Injection volume was set as 10 mL and runtime was
2 min, achieving high sample throughput. LODs and LOQs
were 0.024 and 0.08 mg/mL for TRI, 0.012 and 0.04 mg/mL
for HCT, respectively. Compared with VWD, PDA can pro-
gram wavelength range so that all compounds absorbing in
this wavelength range can be detected in one analysis with-
out having to inject sample multiple times when wavelength
changes.[125] Subramanian et al.[126] developed HPLC-PDA
for the simultaneous detection of TOR and SPI in human
plasma with wavelength range from 235 to 245 nm. Using
Central Composite Design for optimization and after run-
time of 9 min, LODs of TOR and SPI were 25 and
15 ng/mL, and LOQs 80 and 50 ng/mL, respectively. This
showed that HPLC-PDA can be regarded as an effective
alternative to HPLC-MS for routine analysis.
RID is a general-purpose detector of low sensitivity for
most substances. It is not preferred for trace analysis and is
affected by ambient temperature and mobile phase compos-
ition, so not suitable for gradient elutions. It is favorable for
substances with no or weak UV absorptions. Sensitivity is
high especially for sugars detection.[125] Hadjikinova
et al.[82] developed HPLC-RID method for determining sug-
ars like sorbitol in low-calorie dessert foods. Distilled water
was used as mobile phase. Nine analytes were separated after
50 min. LOD and LOQ of sorbitol were 0.17 and
0.56 mg/mL, respectively.
Compared with UVD and RID, FLD is more sensitive
and takes less time for analysis. It possesses selectivity and
only reacts with fluorescent substances or substances that
fluoresce after chemical reactions.[125] Mendia et al.[33] used
UPLC-PDA-FLD for determining 11 drugs including AMI,
FUR and HCT in human plasma. PDA recorded absorption
spectrum between 190 and 400 nm. The optimum excitation
wavelengths (kex) and emission wavelengths (kem) for AMI
were 363 and 415 nm, and those for FUR were 235 and
398 nm. Separation of 11 drugs was achieved in less than
6 min with overall runtime of 8.5 min. LOQs of AMI, FUR
and HCT were 0.08, 0.30 and 0.30 mg/mL, respectively.
Thus, FLD is a favorable choice for diuretics that emit
fluorescence.
3.2. Sensors
Chromatography and spectroscopy dominate in qualitative
and quantitative analysis of drugs because of high sensitivity
and selectivity. In recent years, sensors for diuretics analysis

in biological and pharmaceutical samples have shown
increasing trends. Sensors are fast, accurate, have wide linear
range, simple equipment and low priced which justify their
wider use and recent developments. They are convenient for
analyzing complex samples without prior separations even if
the samples are colored or contain precipitates.[127] The per-
formance of different sensors for analyzing diuretics in real
samples are summarized in Table 3.
3.2.1. Electrochemical sensors
With merits of high sensitivity, rapidity, low cost, simple
device, environmental friendliness, wide application range
and no complex pretreatments, electrochemical sensors are
popular in diuretics analysis. Commonly used electrochem-
ical sensor types are voltametric sensors, potentiometric sen-
sors and amperometric sensors.
Being sensitive, inexpensive, simple and rapid, voltam-
metric sensors are the mostly used electrochemical sensors
in determinations of diuretics in various samples, accounting
for over 90%. Different voltametric techniques such as cyclic
voltammetry (CV),[131] differential pulse voltammetry
(DPV),[136] square wave voltammetry (SWV),[135] and linear
sweep adsorptive stripping voltammetry (LSAdSV) have
already been utilized to realize sensitive and rapid ana-
lysis.[149] Traditionally used working electrodes including
carbon paste electrodes (CPE), glassy carbon electrodes
(GCE), and static mercury drop electrode (SMDE). While
they have some disadvantages which limit their applications,
for example, poor reproducibility and selectivity (CPE), poor
sensitivity and producing toxic waste mercury (SMDE), and
susceptible to contamination by organic and metal com-
pounds (GCE). Novel electrode materials have thus been
synthesized and explored. Various unmodified and chem-
ically modified electrodes have been developed to attain
selective membrane permeability, preferential enrichment,
high selectivity, accelerating electron transfer reactions,
improving sensitivity and stability (Table 3). Examples
include boron-doped diamond electrode (BDD),[3,128,144]
MWCNTs modified CPE (MWCNTs/CPE),[52] amorphous
carbon nitride electrode (a-CNx),[134] reduced graphene
oxide hybrid nanocomposite (rGO/MWCNTs/GCE),[130] and
nickel hydroxide modified nickel electrode.[131]
Said et al.[132] synthesized MnO2 nanoparticles/chitosan-
modified pencil graphite electrode (MnO2 NPs/CS/PGE)
coupled with CV for sensing FUR in human urine. This
electrode had low cost, low background current, ease of
modification of PGE, large surface area, superior electrocata-
lytic activity of MnO2 NPs, good adhesion, biocompatibility,
and low toxicity of chitosan. In BR buffer of pH 3.0, LOD
of 0.00388 lmol/L and linear range of 0.05–4.20 lmol/L
were obtained. Interference studies exhibited superior select-
ivity with over 100-folds excess of citric acid, uric acid, glu-
cose, and metals like Pb2þ and Cu2þ having no interference
with FUR determination (15.0 lmol/L).
Similarly for the improvement of analysis effectiveness,
Wang et al.[137] synthesized C3N4 nanosheets/MOF wrapped
with MIP sensor (C3N4/ZIF-8@MIP/GCE) for the detection
of FUR by DPV (Figure 3C) in human urine and tablets.
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 21
High selectivity and sensitivity could be achieved by ZIF-8
@ MIP through hydrogen bonding and p–p stacking inter-
actions. Increasement in the conductivity and the effective
active surface area of the electrodes were realized by utiliz-
ing GCE coated with C3N4. DPV belongs to pulse techni-
ques and has higher sensitivity than CV. Finally, LOD was
8 nM and linear range was 0.08–100 lM. Thus, it could be a
powerful method for effectively detecting FUR in biological
samples.
For pursuing electrode reproducibility, Kor et al.[133] syn-
thesized FUR-imprinted poly-o-phenylenediamine gold elec-
trode coupled with DPV for the selective determination of
FUR in human urine. In PBS of pH 7.0, 0.07 lmol/L of
LOD and 0.1–7.0 lmol/L of linear range were obtained. The
electrode was stable after a month as it reached 80% of its
original response. RSD was less than 2.9% for the continu-
ous determination of furosemide with seven electrodes, indi-
cating good reproducibility of the electrode.
Potentiometric sensors are mainly ion sensors which con-
sist of ion selective membrane, reference electrodes and
internal standard solution. Conventional solid contact ion
selective electrodes such as GCE have merits of low energy
consumption, portable, nondestructive, and rapid response,
while low reproducibility and stability may occur owing to
the water layer between the applied selective membrane the
solid contact. Thus, Tantawy et al.[142] developed a new
MWCNTs/GCE for potentiometric xipamide determination
to solve the problem of the solid contact systems. They con-
ducted a comparative test between MWCNTs/GCE and bare
GCE, and results showed outstanding properties of this pro-
posed method: LOD of 6.0 lmol/L versus 9.0 lmol/L,
response time of 5 s versus 10 s, lifetime of 30 d versus 25 d,
and potential drift of 0.8 mV/h versus 6 0.8 mV/h. These
changes could be attributed to the hydrophobicity of the
MWCNTs used eliminating the water layer between the
ionic membrane and solid contact.
Compared to potentiometric sensors, amperometric sen-
sors have better anti-interference ability and longer propaga-
tion distance. Multiple pulse amperometry (MPA) is a type
of amperometric technique which allows up to 10 different
potential pulses to be applied and collects the current versus
time relationship for each potential pulse. Flow-injection
analysis (FIA) was often coupled with MPA for drugs ana-
lysis. Compared with traditional methods using voltammet-
ric techniques, FIA-MPA has merits in reducing the
pollution of electrodes, achieving better sensitivity, introduc-
ing automation and higher analytical frequency.[139]
Lourencao et al.[143] used FIA-MPA for detecting HCT in
tablets with BDDE and attained LOD of 0.20 lmol/L, linear
range of 0.40–8.00 lmol/L, and analytical frequency of 89
injections/h. Batch injection analysis (BIA) is an alternate of
FIA which inherits its advantages and has simpler structure
than that of FIA. It is portable and composed of syringes
and batteries. Silva et al.[145] developed BIA-MPA coupled
with BDDE for analyzing FUR and HCT in urine and tab-
lets. They obtained excellent sensitivity (LOD of 0.65 lmol/L
for FUR and 0.63 lmol/L for HCT) and wide linear range
(2–300 lmol/L for FUR and 2–100 lmol/L for HCT) in
Table 3. Sensor methods for diuretics in different matrix.
22

Linear range
Matrix Analytes Working electrode Method Medium (lmol/L) LOD (lmol/L) LOQ (lmol/L) RSD (%) Lifetime Response time Ref
[3]
Tablets HCT BDDE SWV pH 9.0, 29–260 0.08 n.d. 0.91 n.d. n.d.
ammonium
buffer
[128]
Tablets HCT BDDE SWV pH 5.0, BR buffer 2.9–45 0.75 2.5 1.6–1.7 n.d. n.d.
[129]
Tablets ACE SDME SWV pH 2.4, BR buffer 1.98–59.4 2.97 9.92 1.68–1.80 7 d (97.88% n.d.
Y.-J. LIU ET AL.

initial
response)
[130]
Tablets TRI rGO/MWCNTs/GCE DPV pH 7.0, 1.5–120 0.32 n.d. 3.52–3.91 n.d. n.d.
HCT 0.1 mol/L PBS 0.5–120 0.37
[131]
Human urine HCT Nickel hydroxide CV pH 5.6, 0.1 mol/L 13.9–167 7.92 n.d. n.d. n.d. n.d.
modified nickel acetate buffer
electrode
[132]
Human urine FUR MnO2 NPs/CS/PGE CV pH 3.0, BR buffer 0.05–4.20 0.00444 0.01346 2.53 7 d (96.7% initial n.d.
response)
[133]
Human urine FUR FUR-imprinted poly- DPV pH 7.0, 0.0001–0.007 0.00007 n.d. <2.9 30 d (80% initial n.d.
o- 0.2 mol/L PBS response)
phenylenediamine
gold electrode
[134]
Human urine FUR a-CNx SWV pH 4.5, 0.50–99 0.39 n.d. n.d. n.d. n.d.
and tablets 0.04 mol/L BR
buffer
[135]
Human urine HCT MIP/Fe3O4/ edge SWV pH 7.2, PBS 0.025–0.25 0.004 n.d. 2.32 20 d n.d.
and tablets plane pyrolytic 0.25–10 0.013
graphite
[136]
Human urine AMI Portable unmodified DPV pH 4.0, BR buffer 17.76–22.8 0.18 n.d. 2.95 Disposable n.d.
and tablets TRI screen–printed 0.056–3.83 0.0107
electrodes
[137]
Human urine FUR C3N4/ZIF-8@MIP/GCE DPV pH 5.0, BR buffer 0.08–100 0.008 n.d. 1.14 15 d 20 min
and tablets
[138]
Human urine HCT 1-benzyl-4-ferrocenyl- SWV pH 8.0, 0.1–500 0.08 n.d. 1.7–3.4 n.d. n.d.
and tablets 1H-[1,2,3]-triazole 0.1 mol/L PBS
modified glassy
carbon nanotube
electrode
[139]
Synthetic urine HCT Composite electrode FIA- pH 7.5, 10–200 1.3 4.4 3.34 n.d. 128
and tablets made with 75% amperometric 0.04 mol/L BR determinations/h
(w/w) of carbon detector buffer
black and 25%
(w/w) of
poly(ethylene
covinyl)acetate
[140]
Human serum ACE Modified COOH- DPV pH 7.0, 0.1 mol/L 1.0  17.5 0.06 0.2 0.72 n.d. n.d.
CNFs/GCE phosphate
buffer
solutions
(PBS)
[141]
Human serum HCT Tantalum electrode DPV pH 7.0, BR buffer 3–500 1.00 n.d. 0.6 94 d n.d.
and tablets coated with
graphene
nanowalls
[52]
Human blood TOL MWCNT/CPE CV and DPV pH 4.5, 1–7 0.0381 0.127 0.40–1.03 n.d. n.d.
and urine 0.01 mol/L
 phosphate
buffer
[142]
Human plasma Xipamide MWCNT/GCE potentiometric pH 6.0-9.0, BR 10–10000 6 n.d. 1.705 30 d 5s
and tablets measurement buffer
[143]
Tablets HCT Cathodically FIA-MPA 0.05 mol/L H2SO4 0.4–8.0 0.20 n.d. 1.31–2.98 n.d. 89 determinations/h
pretreated-BDD
[144]
Tablets AMI BDDE BIA-MPA pH 10.0, 3.0–160.0 0.13 0.43 0.5–1.1 n.d. 72 determinations/h
FUR 0.04 mol/L BR 12.0– 0.94 mg/L 3.13 mg/L
buffer 160.0 mg/L
[145]
Human urine FUR, HCT BDD BIA-MPA pH 4.0, BR buffer 2–300, 2–100 0.65, 0.63 n.d. <5 n.d. 130
and tablets determinations/h
[146]
Tablets SPI laccase-EPS- SWV pH 7.0, 2.97–28.9 0.94 n.d. n.d. 60 d (86.2% n.d.
MWCNTs/GCE 0.10 mol/L initial
PBS response)
[147]
Human plasma, ACE Binuclear oxo- CV pH 5.6, 0.1 mol/L 0.005–0.025 0.0132 0.0441 <5 500 times n.d.
saliva, and manganese acetate buffer
urine complex based on
oxidase enzyme
[148]
Aqueous FUR Organic-inorganic Fluorescent pH 1–10 0–32 0.551 n.d. n.d. n.d. n.d.
medium hybrid framework sensor
where AuNPs
immobilized onto
organic NPs of
naphthalimide
derivatives
[42]
Human serum ACE Tb3þ-acetylacetone Fluorescent pH 6.8 0.00449–0.128 0.004 0.0121 0.61 2 years n.d.
and tablets photo probe sensor
n.d., not found.
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY
23
24 Y.-J. LIU ET AL.
0.04 mol/L BR buffer (pH 4.0). The analytical frequency was
130 injections/h. By using FIA and BIA, higher analytical
frequency as well as lower reagent cost can be achieved
easily.
3.2.2. Biosensors
Biosensors consist of biosensing elements and signal trans-
ducer, whose recognition element is a biological substance
or a biomimetic substance. Electrochemical biosensors com-
bine biological and electronic detections. Electrode is the
conversion element and fixed carrier, and biologically sensi-
tive substances such as antigens, antibodies, enzymes, hor-
mones, etc. are fixed onto the electrode as sensitive
elements. Laccase-based biosensors have the sensitivity and
selectivity for variety of phenolic compounds such as dopa-
mine and can determine them in complex matrix. In add-
ition, they are low cost, simple manufacture, rapid response,
easy miniaturization, and used for indirect determination of
drugs. Coelho et al.[146] developed laccase-biosensor by
applying MWCNTs and exopolysaccharide botryosphaeran
modified GCE (laccase-EPS-MWCNTs/GCE) coupled with
SWV for the indirect determination of SPI in commercial
pharmaceuticals. SPI reacted with dopamine quinone prod-
uct, formed by the oxidation of dopamine and catalyzed by
laccase on biosensor surface which reduced the peak value
of cathode current and achieved indirect quantification.
LOD of 0.94 lmol/L and linear range of 2.97–28.9 lmol/L
were obtained. 86.2% of initial response was retained and
there was no activity loss after 200 measurements, demon-
strating its stability and lifetime.
Compared to biosensors, bio-inspired sensors have better
catalytic property, sensitivity, and longer life. Machini
et al.[147] applied a bio-inspired sensor using binuclear oxo-
manganese complex based on oxidase enzyme for determin-
ing ACE in urine. LOD was 0.0132 lmol/L and linear range
0.005–0.025 lmol/L. Bio-inspired sensor had good perform-
ance after over 500 times of detections. From the perspective
of economic benefits and analytical efficiency, bio-inspired
sensors can be a favorable alternative to biosensors.
3.2.3. Fluorescent sensors
Electrochemical sensors can achieve low LODs and good
selectivity, however their sizes are relatively large and thus
not easy to carry for on-site measurements. In contrast,
fluorescent sensors have small size, low cost, short response
time, favorable selectivity, and sensitivity. Besides, they are
not affected by external electromagnetic fields. Alexander
et al.[150] synthesized [Zn2(1)2] doped to ethyl cellulose as
fluorescent sensor to detect diuretics in human urine and
obtained LODs of 24 lg/mL for FUR and 43 lg/mL
for TOR.
Gold nanoparticles (AuNPs) are biocompatible, easy to
functionalize and chemically stable for employing in drug
delivery and sensing. AuNPs are coated with organic ligands
for controlling the sensor characteristics. Naphthalimide
derivatives with strong fluorescence, photostability and
adjustability are the suitable choices. Saini et al.[148]
developed an organic-inorganic hybrid framework where
AuNPs immobilized onto organic NPs of naphthalimide
derivatives acted as fluorescent sensor for the selective deter-
mination of FUR in tablets. The synthesized hybrid NPs
were applicable in pH 1–10 and obtained LOD of
0.551 lmol/L and linear range of 0–32 lmol/L, which
showed its potential wide applications in various matrices.
Lanthanide complexes were used as optical sensors for
higher sensitivity, durability and stability. These types of
sensors provided constant signal response for 2 years, and
their stability was improved by 24 times compared with the
service life guaranteed by chromatography and colorim-
etry.[42] Youssef[42] utilized photoprobe of Tb3þ-acetylace-
tone for detecting ACE in tablets and human serum where
LOD and linear range were 0.004 lmol/L and 0.00449–
0.128 lmol/L, respectively. Satisfactory sensitivity and much
longer service life compared with other techniques make
this proposed method an interesting research topic.
3.3. Spectroscopic analysis
Chromatographic methods are prevalent in pharmaceutical
analysis because of favorable sensitivity and selectivity. Most
however require complex sample pretreatments before being
injected to chromatographic systems which cost extra labor,
money, and time. The extraction process may lose some
analytes, making their quantification inaccurate. In contrast,
spectroscopic methods are simple, no extra pretreatments,
convenient to use, low cost, fast, accurate and sensitive.
Besides, there is little or no damage to samples and no pure
sample requirements.[151] Spectrophotometry is the com-
monly used spectral analysis method to detect diuretics in
biological samples or pharmaceutical preparations.[152–154]
Binh et al.[152] developed UV–Vis spectrophotometric
method coupled with Kalman filter algorithm for simultan-
eously analyzing losartan potassium and HCT in tablets.
LOD and LOQ of HCT were 0.037 and 0.12 lg/mL with
RSD <0.167%. The only solvent used in analysis was water.
Compared with previous experiments using HPLC for deter-
mining these two drugs[155] which employed ACN and for-
mic acid as dissolving solvent and LOD and LOQ for HCT
were 0.5 and 1.5 lg/mL, this proposed method proved suit-
able and economical for routine analysis.
Fourier transform infrared spectroscopy has high reso-
lution. It is an accurate, rapid, simple, low cost, sensitive
and time-saving analysis method. It was first utilized by
Chandarana et al.[153] in 2019 for determining diuretics in
tablets. The following results were obtained: LOD,
0.016 lg/mL; LOQ, 0.050 lg/mL; RSD, <0.1661%; accuracy,
98–102%. Thus, it also can be used as a routine method for
analysis of diuretics with low cost. However, the spectral
interference caused by spectral lines overlaps may seriously
compromise the interpretations and the selectivity.
Phase-resolved fluorescence spectroscopy (PRFS) has
been developed to solve the problems of simultaneous meas-
urements of two or more compounds in the matrix with
overlapping excitation/emission curves, and increase the
selectivity and sensitivity. The analysis requirement is to

know the steady-state spectrum of individual components
and to suppress the exact phase angle of each constituent in
the mixture. This is based on eliminating the contribution
of one constituent in total spectrum of the mixture.[156] The
combination of wavelength selectivity and fluorescence
selectivity can be achieved by appropriate combination of
wavelength and detector phase angle which improves the
method selectivity. Sanchez et al.[154] utilized PRFS for the
selective determination of FUR and TRI with overlapping
excitation/emission curves in binary samples. LODs for FUR
and TRI were 3.04 lg/mL and 9.0 ng/mL (RSD < 8%),
respectively. Without information provided by the whole
spectrum of samples and complex separation techniques,
this proposed method allowed a resolution at lg/mL level
for the mixture and got satisfactory results.
Conventional fluorescence spectroscopic methods based
on single-wavelength excitation or single-wavelength emis-
sion prove insufficient for analyzing several compounds in
complex matrices such as biological samples. Excitation-
emission matrix fluorescence (EEMF) also called fluores-
cence fingerprinting or three-dimensional fluorescence can
simultaneously collect fluorescence data in different excita-
tion and emission wavelength ranges, making fluorescence
analysis more reliable and accurate.[157] Hu et al.[158] used
EEMF combined with chemometrics for determining AMI
and TRI whose peaks overlapped in human urine and
plasma, which required few sample pretreatments and
achieved precise qualitative and quantitative information for
both. Optimum EEMF conditions were: kex ¼ 240–430 nm
and kem ¼ 370–530 nm in 2 nm steps, 30,000 nm/min scan-
ning rate (4 min per sample), and 650 V detector voltage.
LODs of AMI and TRI were 0.43 and 0.02 ng/mL and LOQs
were 1.29 and 0.07 ng/mL for human urine; LODs were 0.9
and 0.12 ng/mL and LOQs were 2.74 and 0.35 ng/mL,
respectively, for human plasma. Thus, by applying EEMF
and chemometrics, highly sensitive results could be obtained
for diuretics in complex biological samples.
3.4. Other analytical methods
3.4.1. GC–MS
GC–MS is the sensitive method for determining volatile sub-
stances or volatile substances after derivatizations. Owing to
the nonvolatile property of diuretics, derivative reagents are
usually in need, such as ethanethiol/NH4I/N-Methyl-N-(tri-
methylsilyl) trifluoroacetamide (MSTFA),[159]
[160]
MSTFA/ethanethiol/NH4I/dodecane and pentafluoroben-
zyl bromide (PFB-Br).[161] Compare to LC-MS, it gets rid of
limitations of organic solvents as mobile phase, thus is more
environmental. According to WADA requirements, anti-
doping laboratories should adopt two analytical methods,
LC-MS, and GC-MS, for the initial testing procedures. They
together cover all abused drugs in prohibited list.[160] GC
columns include capillary column and packed column.
Macherey-Nagel Optima 5MS Accent (30 m  0.25 mm
i.d.)[162] and Macherey-Nagel fused-silica capillary column
Optima 17 (15 m  0.25 mm i.d.)[161] have been used for
separating diuretics with favorable efficiency. Lately, Agilent
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 25
HP ultra 1 column (15 m  0.20 mm i.d.), consisting of two
parts, had been utilized for this purpose. The first part (5 m)
was from inlet to back flush unit and second part (10 m)
from back flush unit to the detector. The installed backwash
system could remove pollutants accumulated during the first
part of GC operation to the inlet instead of ion source at
the last stage of GC operation, thus extending the life of
chromatographic column. Since the first part can be used as
precolumn at the same time (instead of complete 15 m col-
umn), it can easily be substituted which minimizes the
maintenance cost.[160] Polet et al.[160] used this column for
low energy electron impact GC-Q-TOF-MS for screening
doping substances and after 14.1 min of single run, 294 tar-
get compounds (including diuretics) were detected.
GC-MS often utilizes electron impact ionization (EI) or
chemical ionization (CI) which is different from LC-MS that
commonly uses ESI or APCI as ionization sources. EI belongs
to “hard ionization” and is nonselective ionization. It has high
ionization efficiency and sensitivity. Albertsdottir et al.[159]
applied GC-Q-TOF-EI-MS for screening 263 doping substan-
ces in human urine. Derivatization mixture consisted of
400 lL ethanethiol, 200 mg NH4I and 10 mL N-Methyl-N-(tri-
methylsilyl) trifluoroacetamide. After 14.8 min runtime, the
LODs of diuretics were between 2 and 40 ng/mL. The applica-
tion of HRMS makes open screening possible owing to the
traceability and compatibility with the library of the collection
data by FS mode.[163] This undoubtedly broadens the practical
application range of GC-MS. EI is not applicable to samples
that are difficult to volatilize and thermally unstable. EI
method can only detect positive ions but not negative ions,
besides, due to massive fragmentation caused by EI, molecular
ions of many compounds cannot be observed. CI is “soft ion-
ization” as an alternative to avoid major fragmentations.[117]
Begou et al.[161] applied stable-isotope dilution GC-NICI-MS
method for determining ACE in human urine. After 50 mL
urine was evaporated to dryness, ACE and 250 mM IS d3ACE
were base catalyzed in 30% pentafluorobenzyl bromide in
ACE presence (1 h, 30  C), and recombined in 200 mL toluene.
LOD and LOQ for ACE were 0.3 pmol and 25 mM. By com-
parison, LC methods which provides no derivatization pro-
cedure have simpler operations than GC methods. While in
cases when an unknown endogenous compound in biological
samples is co-eluted with the analyte, LC methods become
inaccurate in the lower mM range. Thus, GC methods with
derivatization prove a favorable alternative.
3.4.2. TLC
TLC is a chromatographic separation technique using solid
media coated onto the support plate as stationary phase and
appropriate solvent as mobile phase to separate, identify and
quantify the components in the mixture sample. This tech-
nique is widely used and suitable for routine pharmaceutical
analysis because of convenient operation, simple equipment,
low cost, easy separation of mixture, wide selection range of
mobile phases, flexible sample differentiation, and high sam-
ple loading capacity.[125] Dołowy et al.[164] used TLC-densi-
tometry for determining SPI in bulk drugs and
pharmaceutical formulations. The selected and optimized
26 Y.-J. LIU ET AL.
TLC system was chromatographic plate precoated with mix-
ture of silica gel 60, kieselguhr F254, and n-hexane-ethyl
acetate-glacial acetic acid (24.5: 24.5: 1, v/v/v) as mobile
phase. LOD and LOQ of SPI were 0.034 and 0.103 lg/spot,
respectively, showing enough sensitivity for identification
and quantification of SPI.
High performance thin layer chromatography (HPTLC)
has been developed to further improve the sensitivity and
resolution of TLC. The developed HPTLC provides high
separation efficiency by aligning to HPLC using uniform
fine particle stationary phase. Compared with TLC, the
detection sensitivity of HPTLC is increased and analysis
time is shortened from 20–200 min to 3–20 min through
programed multi-level expansion or hierarchical and bidirec-
tional expansion technology.[125] Compared with HPLC,
HPTLC is simple, cheap and allows larger number of sam-
ples in a run (up to 36 sample tracks per plate),[165] which
is more applicable to routine analysis. Naguib et al.[84] uti-
lized HPTLC-UVD for analyzing FUR, SPI and CAN in
human plasma, tablets, and pure forms. The chromato-
graphic conditions were silica gel HPTLC F254 plates using
ethyl acetate: triethylamine: acetic acid (9: 0.7: 0.5, v/v/v).
The detection wavelength was set to 254 nm. LOQs of FUR,
SPI and CAN were 0.195, 0.048, and 0.045 mg/mL, and
LODs were 0.059, 0.015, and 0.014 mg/mL, respectively.
Simpler operations, less expensive equipment and acceptable
sensitivity make HPTLC-UVD a routine alternative analysis
for diuretics.
3.4.3. CE
HPLC is the primary analytical method recommended by
pharmacopoeias for determining the drug impurities. CE
however has also been proved as a vital alternative method.
Compared with the HPLC application, CE requires lower
sample volumes and solvent consumptions, shorter separ-
ation times, lower operation costs and no complex sample
pretreatments. Besides, CE provides higher resolving power
than HPLC.[166] There are many separation modes in CE.
The most often used are capillary zone electrophoresis
(CZE), capillary gel electrophoresis and micellar electro-
kinetic chromatography (MEKC). CZE is the basic and
widely used separation mode in CE analysis. It has high effi-
ciency, fast speed, trace amount detectability, and especially
suitable for separating charged compounds. Li et al.[166]
applied CZE-PDA for the rapid determination of SPI and
CAN in pharmaceutical formulations and human urine.
Under optimum conditions: 20 mM PBS containing 4.5 g/L
sulfated-b-cyclodextrin (pH 5.5), 15 kV, 25  C and 260 nm
wavelength; LOD of 0.56 and 0.20 lg/mL were obtained for
SPI and CAN, and LOQ were 1.87 and 0.67 lg/mL, respect-
ively. MEKC can simultaneously separate neutral and ionic
substances which broadens the application scope of CE. By
adding ionic surfactants such as SDS to buffer to form
micelles, the separated substances are distributed between
aqueous phase and micellar phase and migrate in the capil-
lary with electroosmosis for achieving the best of separation.
Fayez et al.[167] applied MEKC-PDA to separate and deter-
mine HCT in tablets containing co-formulated drugs and
major impurity or HCT degradation products (chlorothia-
zide, similar structure as HCT). Optimum conditions were:
17 mM borate buffer (pH 9), 5.2 mM SDS, 12 kV and 25  C.
Less than 8 min was required for overall separation. LOD
and LOQ of HCT were 1.54 and 4.67 lg/mL, respectively.
Linearity was between 5.00 and 100.00 lg/mL. MEKC chro-
matogram exhibited satisfactory separation for bisoprolol,
chlorothiazide, HCT, and irbesartan with retention time at
5.55, 6.86, 7.36, and 7.84 min, respectively.
3.5. Summary of determination techniques
Among all analytical techniques for determining diuretics in
biological samples and pharmaceutical formulations, HPLC
coupled with detectors such as MS, UVD, RID and FLD are
the most prevalent owing to superior separation perform-
ance of HPLC and favorable sensitivity of these detectors.
HPLC-MS/MS is effective due to excellent qualitative and
quantitative capabilities of MS. In recent years, the applica-
tion of HRMS for targeted and untargeted analysis has
screened multiple diuretics in complex matrices.
Chromatography-based methods achieve high sensitivity and
selectivity, they are however relatively expensive and need
complex sample pretreatments. In contrast, spectrometric
methods are cheaper, simpler, and have acceptable sensitiv-
ity and selectivity. Electrochemical methods have been devel-
oped as simple, rapid, low cost, and with excellent analytical
performance. In summary, for detecting diuretics in athletes
of sports events and to facilitate the on-site detection, the
development of portable, rapid, sensitive, selective, and
environment friendly detection methods is the top priority.
For detecting and controlling the diuretics in pharmaceutical
formulations, HPLC-MS or UVD occupies the primary
place, however various spectroscopic and electrochemical
sensor methods are also being developed.
4. Conclusion
Diuretics are the common drugs in clinical treatments of
edema, hypertension, and other diseases. The drugs dosages
need accuracy to avoid health damages. Monitoring the clin-
ical blood drug concentrations and quality control of
pharmaceutical formulations have always been in focus. In
addition, accurate and rapid urine tests before competitions
are ensured for the athletes’ fair competitions. Such moni-
toring urges developments of various detection technologies.
This review has summarized and compared the pretreatment
and detection techniques of diuretics since 2015.
Regarding pretreatment methods for extraction of diu-
retics from biological samples, techniques such as UAE,
SPE, LLE, LPME, and SPME have been applied. SPE has
been frequently used with the development of novel adsorb-
ent materials, for instance, MNPs, MWCNTs, and MIPs
which provide desired extraction efficiencies. Besides, the
automation of SPE brings accuracy, reproducibility, and
avoids tedious steps of sample handlings. SPME is being
rapidly developed and adhere to the concept of green chem-
istry. It requires less quantities of samples to meet the

human sample collection protocols. Various types of SPME
such as TF-SPME, SBME, and BAlE are developed for
extracting diuretics of diverse physicochemical properties.
The pretreatment techniques are developed and optimized
to achieve rapidity, simple device, ease of operation, auto-
mation, low consumption of organic solvents and samples,
and convenience for on-site analysis.
For diuretics determinations, HPLC-MS is the preferred
and common method because of its superior separation effi-
ciency and excellent qualitative and quantitative ability. The
application of HRMS screens and analyzes known and
unknown substances in complex matrices. Besides, electro-
chemical sensor methods play role in detecting diuretics. It
takes few seconds to complete the accurate and automated
determinations. A variety of materials modified electrodes
are fabricated to achieve higher sensitivity and selectivity.
For detecting diuretics in biological and pharmaceutical
formulations, the use of rapid, accurate, convenient, eco-
friendly and sensitive methods with less sample consump-
tion will be the developmental focus in future.
CRediT authorship contribution statement
Ya-jie Liu: Writing –review & editing. Yu Bian: Writing
–review & editing. Yuan Zhang: Writing –review & editing.
Yi-xin Zhang: Supervision. Ai Ren: Supervision. Shu-han
Lin: Editing. Xue-song FENG: Conceptualization, Validation.
Xin-yuan Zhang: Conceptualization, Validation.
Disclosure statement
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Funding
This work was supported by the Scientific Research Project of the
Department of Education of Liaoning Province (ZF2019036).
ORCID
Xue-song Feng http://orcid.org/0000-0002-6981-5289
References
[1] Hoorn, E. J.; Ellison, D. H. Diuretic Resistance. Am. J. Kidney
Dis. 2017, 69, 136–142. DOI: 10.1053/j.ajkd.2016.08.027.
[2] Sarafidis, P. A.; Georgianos, P. I.; Lasaridis, A. N. Diuretics in
Clinical Practice. Part I: mechanisms of Action,
Pharmacological Effects and Clinical Indications of Diuretic
Compounds. Expert Opin. Drug Saf. 2010, 9, 243–257. DOI:
10.1517/14740330903499240.
[3] Moraes, J. T.; Salamanca-Neto, C. A. R.; Svorc, L'.; Sartori,
E. R. Advanced Sensing Performance towards Simultaneous
Determination of Quaternary Mixture of Antihypertensives
Using Boron-Doped Diamond Electrode. Microchem. J. 2017,
134, 173–180. DOI: 10.1016/j.microc.2017.06.001.
[4] De Wilde, L.; Roels, K.; Polet, M.; Van Eenoo, P.; Deventer, K.
Identification and Confirmation of Diuretics and Masking
Agents in Urine by Turbulent Flow Online Solid-Phase
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 27
Extraction Coupled with Liquid Chromatography-Triple
Quadrupole Mass Spectrometry for Doping Control. J.
Chromatogr. A 2018, 1579, 31–40. DOI: 10.1016/j.chroma.
2018.10.032.
[5] Nan, P. Detection of Diuretic Doping by Capillary
Electrophoresis and Electrochemical Technology: A Mini-
Review. CPA. 2022, 18, 34–42. DOI: 10.2174/15734129179992
01217163607.
[6] Tao, Y.; Wang, S.; Wang, L.; Song, M.; Hang, T. Simultaneous
Determination of Indapamide, Perindopril and Perindoprilat
in Human Plasma or Whole Blood by UPLC-MS/MS and Its
Pharmacokinetic Application. J. Pharm. Anal. 2018, 8, 333–
340. DOI: 10.1016/j.jpha.2018.05.004.
[7] Hoshikawa, K.; Naito, T.; Saotome, M.; Maekawa, Y.;
Kawakami, J. Validated Liquid Chromatography Coupled to
Tandem Mass Spectrometry Method for Simultaneous
Quantitation of Tolvaptan and Its Five Major Metabolites in
Human Plasma. Ann. Clin. Biochem. 2019, 56, 387–396. DOI:
10.1177/0004563219827045.
[8] Zhang, L.; Wang, R.; Tian, Y.; Zhang, Z. Determination of
Torasemide in Human Plasma and Its Bioequivalence Study
by High-Performance Liquid Chromatography with
Electrospray Ionization Tandem Mass Spectrometry. J. Pharm.
Anal. 2016, 6, 95–102. DOI: 10.1016/j.jpha.2015.11.002.
[9] Vojta, J.; Jedlicka, A.; Coufal, P.; Janeckova, L. A New, Rapid,
Stability-Indicating UPLC Method for Separation and
Determination of Impurities in Amlodipine Besylate, Valsartan
and Hydrochlorothiazide in Their Combined Tablet Dosage
Form. J. Pharm. Biomed. Anal. 2015, 109, 36–44. DOI: 10.
1016/j.jpba.2015.01.059.
[10] Kumar, A.; Dwivedi, S. P.; Prasad, T. Method Validation for
Simultaneous Quantification of Olmesartan and
Hydrochlorothiazide in Human Plasma Using LC-MS/MS and
Its Application through Bioequivalence Study in Healthy
Volunteers. Front. Pharmacol. 2019, 10, 810. DOI: 10.3389/
fphar.2019.00810.
[11] Patel, J. R.; Pethani, T. M.; Vachhani, A. N.; Sheth, N. R.;
Dudhrejiya, A. V. Development and Validation of
Bioanalytical Method for Simultaneous Estimation of Ramipril
and Hydrochlorothiazide in Human Plasma Using Liquid
Chromatography-Tandem Mass Spectrometry. J. Chromatogr.
B Analyt. Technol. Biomed. Life Sci. 2014, 970, 53–59. DOI:
10.1016/j.jchromb.2014.08.023.
[12] Moola, K. S.; Challa, B. S. R.; Bannoth, C. K. Quantification of
Tolvaptan in Rabbit Plasma by LC-MS/MS: Application to a
Pharmacokinetic Study. J. Pharm. Anal. 2015, 5, 371–377.
DOI: 10.1016/j.jpha.2014.09.001.
[13] Ramakrishna, R.; Puttrevu, S. K.; Bhateria, M.; Bala, V.;
Sharma, V. L.; Bhatta, R. S. Simultaneous Determination of
Azilsartan and Chlorthalidone in Rat and Human Plasma by
Liquid Chromatography-Electrospray Tandem Mass
Spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life
Sci. 2015, 990, 185–197. DOI: 10.1016/j.jchromb.2015.03.018.
[14] Marube, L. C.; Caldas, S. S.; Soares, K. L.; Primel, E. G.
Dispersive Liquid-Liquid Microextraction with Solidification of
Floating Organic Droplets for Simultaneous Extraction of
Pesticides, Pharmaceuticals and Personal Care Products.
Microchim. Acta 2015, 182, 1765–1774. DOI: 10.1007/s00604-
015-1507-7.
[15] Ahmad Panahi, H.; Ejlali, M.; Chabouk, M. Two-Phase and
Three-Phase Liquid-Phase Microextraction of
Hydrochlorothiazide and Triamterene in Urine Samples.
Biomed. Chromatogr. 2016, 30, 1022–1028. DOI: 10.1002/bmc.
3645.
[16] Ho, T.-T.; Li, Z.-G.; Lin, H.-Y.; Lee, M.-R. Determination of
Diuretics in Urine Using Immobilized Multi-Walled Carbon
Nanotubes in Hollow Fiber Liquid-Phase Microextraction
Combined with Liquid Chromatography-Tandem Mass
Spectrometry. J. Chinese Chemical Soc. 2013, 60, 1033–1042.
DOI: 10.1002/jccs.201200603.
28 Y.-J. LIU ET AL.
[17] Wang, X. H.; Xie, L. F.; Dong, Q.; Liu, H. L.; Huang, Y. P.;
Liu, Z. S. Synthesis of Monodisperse Molecularly Imprinted
Microspheres with Multi-Recognition Ability via Precipitation
Polymerization for the Selective Extraction of Cyromazine,
Melamine, Triamterene and Trimethoprim. J. Chromatogr. B
Analyt. Technol. Biomed. Life Sci. 2015, 1007, 127–131. DOI:
10.1016/j.jchromb.2015.11.009.
[18] Yilmaz, H.; Basan, H. Preconcentration of Indapamide from
Human Urine Using Molecularly Imprinted Solid-Phase
Extraction. J. Sep. Sci. 2015, 38, 3090–3095. DOI: 10.1002/jssc.
201500427.
[19] Liu, M.; Lv, B.; Jiang, H.; Yuan, P.; Zhu, H.; Gao, B.
Determination of Diuretics in Human Urine Using HPLC
Coupled with Magnetic Solid-Phase Extraction Based on a
Metal-Organic Framework. Biomed. Chromatogr 2020, 34,
e4876.
[20] Bagheri, H.; Khanipour, P.; Asgari, S. Magnetic Field Assisted
mu-Solid Phase Extraction of anti-Inflammatory and Loop
Diuretic Drugs by Modified Polybutylene Terephthalate
Nanofibers. Anal. Chim. Acta. 2016, 934, 88–97. DOI: 10.
1016/j.aca.2016.06.003.
[21] Arabi, M.; Ghaedi, M.; Ostovan, A. Development of a Lower
Toxic Approach Based on Green Synthesis of Water-
Compatible Molecularly Imprinted Nanoparticles for the
Extraction of Hydrochlorothiazide from Human Urine. ACS
Sustain. Chem. Eng. 2017, 5, 3775–3785. DOI: 10.1021/acssu-
schemeng.6b02615.
[22] Almeida, C.; Ahmad, S. M.; Nogueira, J. M. Bar Adsorptive
Microextraction Technique – Application for the
Determination of Pharmaceuticals in Real Matrices. Anal.
Bioanal. Chem. 2017, 409, 2093–2106. DOI: 10.1007/s00216-
016-0156-y.
[23] Al-Hashimi, N. N.; Aleih, H. A.; Al-Zoubi, N. M.; Hamed,
S. H. Solid Bar Microextraction and HPLC/DAD
Determination of Diuretic Drugs and Its Application to
Spiked Human Urine. Jordan J. Pharmaceut. Sci. 2018, 11, 39–
53.
[24] Zheng, X.; Gao, H.; Cui, X.; Zhang, Y.; Chen, R.; Wang, Y.;
Lauruol, P.; Wang, H. Simultaneous Determination of
Indapamide, Perindopril and Its Active Metabolite
Perindoprilat in Human Plasma Using UPLC-MS/MS Method.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2021, 1169,
122585. DOI: 10.1016/j.jchromb.2021.122585.
[25] Pandya, J. J.; Bhatt, N. M.; Chavada, V. D.; Sharma, P.; Sanyal,
M.; Shrivastav, P. S. Simultaneous Analysis of Aliskiren and
Hydrochlorothiazide in Pharmaceutical Preparations and
Spiked Human Plasma by HPTLC. J. Taibah Univ. Sci. 2017,
11, 667–676. DOI: 10.1016/j.jtusci.2016.05.001.
[26] Shah, P. A.; Sharma, P.; Shah, J. V.; Sanyal, M.; Shrivastav,
P. S. Simultaneous Analysis of Losartan, Its Active Metabolite,
and Hydrochlorothiazide in Human Plasma by a UPLC-
MS/MS Method. Turk. J. Chem. 2015, 39, 714–733. DOI: 10.
3906/kim-1502-4.
[27] Shah, J. V.; Shah, P. A.; Shah, P. V.; Sanyal, M.; Shrivastav,
P. S. Fast and Sensitive LC-MS/MS Method for the
Simultaneous Determination of Lisinopril and
Hydrochlorothiazide in Human Plasma. J. Pharm. Anal. 2017,
7, 163–169. DOI: 10.1016/j.jpha.2016.11.004.
[28] Shah, J. V.; Shah, P. A.; Sanyal, M.; Shrivastav, P. S.
Simultaneous Quantification of Amiloride and
Hydrochlorothiazide in Human Plasma by Liquid
Chromatography-Tandem Mass Spectrometry. J. Pharm. Anal.
2017, 7, 288–296. DOI: 10.1016/j.jpha.2017.03.007.
[29] Patel, B.; Jangid, A. G.; Suhagia, B. N.; Desai, N. Challenges in
Simultaneous Determination of Hydrochlorothiazide and
Ramipril in Human Plasma: Application to a Bioequivalence
Study. J. Chromatogr. Sci. 2018, 56, 867–878. DOI: 10.1093/
chromsci/bmy055.
[30] Palakeeti, B.; Rao, P. N.; Chinta, J. P. Development of New
Stability Indicating UPLC-UV Method for the Extraction and
Quantification of Perindopril and Indapamide from Human
Plasma. Futur. J. Pharm. Sci. 2021, 7, 77. DOI: 10.1186/
s43094-021-00220-8.
[31] Pandya, J. J.; Sanyal, M.; Shrivastav, P. S. Simultaneous
Densitometric Analysis of Amlodipine, Hydrochlorothiazide,
Lisinopril, and Valsartan by HPTLC in Pharmaceutical
Formulations and Human Plasma. J. Liquid Chromatogr.
Related Technol. 2017, 40, 467–478. DOI: 10.1080/10826076.
2017.1324482.
[32] Jiang, J.; Tian, L.; Huang, Y.; Yan, Y.; Li, Y. A Rapid and
Sensitive LC-MS/MS-ESI Method for the Determination of
Tolvaptan and Its Two Main Metabolites in Human Plasma. J.
Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2016, 1027,
158–164. DOI: 10.1016/j.jchromb.2016.03.032.
[33] Gonzalez Mendia, O.; Blanco, M. E.; Rico, E.; Alonso, M. L.;
Maguregui, M. I.; Alonso, R. M. Efficient Method
Development and Validation for the Determination of
Cardiovascular Drugs in Human Plasma by SPE–UHPLC–
PDA–FLD. Chromatographia 2017, 80, 605–615. DOI: 10.
1007/s10337-017-3274-6.
[34] ALOthman, A. L.; Alsheetan, K. M.; Aboul-Enein, H. Y.; Ali,
I. Applications of Shun Shell Column and Nanocomposite
Sorbent for Analysis of Eleven anti-Hypertensive in Human
Plasma. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
2020, 1146, 122125. DOI: 10.1016/j.jchromb.2020.122125.
[35] Chen, X.; Teng, W.; Miao, L.; Wu, Y.; Chen, D.; Huang, L.;
Pan, J.; Wang, N.; Fang, J.; Liang, Y. Simultaneous Analysis of
Hydrochlorothiazide, Triamterene and Reserpine in Rat
Plasma by HPLC and DSPE. Chromatographia 2016, 79, 451–
456. DOI: 10.1007/s10337-016-3060-x.
[36] Wang, X. H.; Zhang, J.; Peng, C.; Dong, Q.; Huang, Y. P.; Liu,
Z. S. Comparison of Multi-Recognition Molecularly Imprinted
Polymers for Recognition of Melamine, Cyromazine,
Triamterene, and Trimethoprim. Anal. Bioanal. Chem. 2015,
407, 7145–7155. DOI: 10.1007/s00216-015-8878-9.
[37] Elfadil, D.; Palmieri, S.; Della Pelle, F.; Sergi, M.; Amine, A.;
Compagnone, D. Enzyme Inhibition Coupled to Molecularly
Imprinted Polymers for Acetazolamide Determination in
Biological Samples. Talanta 2022, 240, 123195. DOI: 10.1016/j.
talanta.2021.123195.
[38] Zeng, L.; Li, Y.; Wu, X.; Zhang, J.; Xie, J.; Sun, C.
Simultaneous Determination of 10 Adulterants in
Antihypertensive Functional Foods Using Multi-Walled
Carbon Nanotubes-Dispersive Solid-Phase Extraction Coupled
with High Performance Liquid Chromatography. J.
Chromatogr. Sci. 2015, 53, 1611–1621. DOI: 10.1093/chromsci/
bmv031.
[39] Wang, Y.; Jie, Y.; Hu, Q.; Yang, Y.; Ye, Y.; Zou, S.; Xu, J.;
Ouyang, G. A Polymeric Solid-Phase Microextraction Fiber for
the Detection of Pharmaceuticals in Water Samples. J.
Chromatogr. A 2020, 1623, 461171. DOI: 10.1016/j.chroma.
2020.461171.
[40] Chen, D.; Xu, Q.; Lu, Y.; Mao, Y.; Yang, Y.; Tu, F.; Xu, J.;
Chen, Y.; Jiang, X.; Lu, J.; Yang, Z. The QuEChERS Method
Coupled with High-Performance Liquid Chromatography-
Tandem Mass Spectrometry for the Determination of
Diuretics in Animal-Derived Foods. J. Food Compos. Anal.
2021, 101, 103965. DOI: 10.1016/j.jfca.2021.103965.
[41] Helfer, A. G.; Michely, J. A.; Weber, A. A.; Meyer, M. R.;
Maurer, H. H. Orbitrap Technology for Comprehensive
Metabolite-Based Liquid Chromatographic-High Resolution-
Tandem Mass Spectrometric Urine Drug Screening -
Exemplified for Cardiovascular Drugs. Anal. Chim. Acta. 2015,
891, 221–233. DOI: 10.1016/j.aca.2015.08.018.
[42] Youssef, A. O. A Highly Selective and Sensitive Tb(3þ)-
Acetylacetone Photo Probe for the Assessment of
Acetazolamide in Pharmaceutical and Serum Samples.
Spectrochim. Acta. A Mol. Biomol. Spectrosc. 2018, 195, 47–52.
DOI: 10.1016/j.saa.2018.01.047.

[43] Muller, L. S.; Moreira, A. P. L.; Muratt, D. T.; Viana, C.; de
Carvalho, L. M. An Ultra-High Performance Liquid
Chromatography-Electrospray Tandem Mass Spectrometric
Method for Screening and Simultaneous Determination of
Anorexic, Anxiolytic, Antidepressant, Diuretic, Laxative and
Stimulant Drugs in Dietary Supplements Marketed for Weight
Loss. J. Chromatogr. Sci. 2019, 57, 528–540. DOI: 10.1093/
chromsci/bmz025.
[44] Vinatoru, M.; Mason, T. J.; Calinescu, I. Ultrasonically
Assisted Extraction (UAE) and Microwave Assisted Extraction
(MAE) of Functional Compounds from Plant Materials. TrAC,
Trends Anal. Chem. 2017, 97, 159–178. DOI: 10.1016/j.trac.
2017.09.002.
[45] Huerta, B.; Jakimska, A.; Llorca, M.; Ruhi, A.; Margoutidis, G.;
Acuna, V.; Sabater, S.; Rodriguez-Mozaz, S.; Barcelo, D.
Development of an Extraction and Purification Method for the
Determination of Multi-Class Pharmaceuticals and Endocrine
Disruptors in Freshwater Invertebrates. Talanta 2015, 132,
373–381. DOI: 10.1016/j.talanta.2014.09.017.
[46] Li, W.; Jian, W.; Fu, Y. Basic Sample Preparation Techniques
in LC-MS Bioanalysis: Protein Precipitation, Liquid–Liquid
Extraction, and Solid-Phase Extraction. Sample Prep. LC-MS
Bioanal. 2019, 1, 1–30. DOI: 10.1002/9781119274315.
[47] Elkady, E. F.; Mandour, A. A.; Algethami, F. K.; Aboelwafa,
A. A.; Farouk, F. Sequential Liquid-Liquid Extraction Coupled
to LC-MS/MS for Simultaneous Determination of Amlodipine,
Olmesartan and Hydrochlorothiazide in Plasma Samples:
Application to Pharmacokinetic Studies. Microchem. J. 2020,
155, 104757. DOI: 10.1016/j.microc.2020.104757.
[48] Kojro, G.; Wroczy nski, P. Cloud Point Extraction in the
Determination of Drugs in Biological Matrices. J. Chromatogr.
Sci. 2020, 58, 151–162. DOI: 10.1093/chromsci/bmz064.
[49] Mahdi, M. I.; Kadhim, K. H. Novel Approach and Cloud
Point Extraction Method for Determination of Acetazolamide
Drug. Indian J. Forensic Med. Toxicol. 2020, 14(2), 586–592.
DOI: 10.37506/ijfmt.v14i2.2915
[50] Jeannot, M. A.; Cantwell, F. F. Solvent Microextraction into a
Single Drop. Anal. Chem. 1996, 68, 2236–2240. DOI: 10.1021/
ac960042z.
 Ramos-Payan, M.; Oca~
[51] Bello-Lopez, M. A.; na-Gonzalez, J. A.;
Fernandez-Torres, R.; Callej on-Moch on, M. Analytical
Applications of Hollow Fiber Liquid Phase Microextraction
(HF-LPME): A Review. Anal. Lett. 2012, 45, 804–830. DOI:
10.1080/00032719.2012.655676.
[52] Pandit, U. J.; Khan, I.; Wankar, S.; Raj, K. K.; Limaye, S. N.
Development of Electrochemical Method for Determination of
Tolvaptan at MWCNT/CPE in Pharmaceutical Preparations
and Human Biological Fluids. Anal. Chem. Lett. 2015, 5, 338–
350. DOI: 10.1080/22297928.2016.1140073.
[53] Varshney, K. Carbon Nanotubes: A Review on Synthesis,
Properties and Applications. Int. J. Eng. Res. General Sci. 2014,
2, 660.
[54] Saraji, M.; Boroujeni, M. K. Recent Developments in
Dispersive Liquid-Liquid Microextraction. Anal. Bioanal.
Chem. 2014, 406, 2027–2066. DOI: 10.1007/s00216-013-7467-z.
[55] Han, D.; Tang, B.; Row, K. H. Determination of Diuretic
Drugs in Human Urine Using Dispersive Liquid–Liquid
Microextraction by High Performance Liquid
Chromatography. J. Liquid Chromatogr. Related Technol. 2013,
36, 2069–2081. DOI: 10.1080/10826076.2012.712931.
[56] Ghambarian, M.; Yamini, Y.; Esrafili, A. Liquid-Phase
Microextraction Based on Solidified Floating Drops of Organic
Solvents. Microchim. Acta 2013, 180, 519–535. DOI: 10.1007/
s00604-013-0969-8.
[57] Moradi, M.; Yamini, Y.; Ebrahimpour, B. Emulsion-Based
Liquid-Phase Microextraction: A Review. J. Iran. Chem. Soc.
2014, 11, 1087–1101. DOI: 10.1007/s13738-013-0376-4.
[58] Regueiro, J.; Llompart, M.; Garcia-Jares, C.; Garcia-
Monteagudo, J. C.; Cela, R. Ultrasound-Assisted
Emulsification-Microextraction of Emergent Contaminants
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 29
and Pesticides in Environmental Waters. J. Chromatogr. A
2008, 1190, 27–38. DOI: 10.1016/j.chroma.2008.02.091.
[59] Ramkumar, A.; Ponnusamy, V. K.; Jen, J.-F. Rapid
Determination of Indapamide in Human Urine Using Novel
Low-Density Solvent Based Ultrasound Assisted Emulsification
Microextraction Coupled with High Performance Liquid
Chromatography-Variable Wavelength Detection. Anal.
Methods 2013, 5, 2572. DOI: 10.1039/c3ay40187a.
[60] Bian, Y.; Zhang, Y.; Zhou, Y.; Li, G.-H.; Feng, X.-S. Progress
in the Pretreatment and Analysis of Flavonoids: An Update
since 2013. Sep. Purif. Rev. 2022, 51, 11–37. DOI: 10.1080/
15422119.2020.1801469.
[61] Mahrouse, M. A. Simultaneous Ultraperformance Liquid
Chromatography/Tandem Mass Spectrometry Determination
of Four Antihypertensive Drugs in Human Plasma Using
Hydrophilic-Lipophilic Balanced Reversed-Phase Sorbents
Sample Preparation Protocol. Biomed. Chromatogr. 2018, 32,
e4362. DOI: 10.1002/bmc.4362.
[62] Hudari, F. F.; Zanoni, M. V. B. A Glassy Carbon Electrode
Modified with Reduced Graphene Oxide for Sensitive
Determination of Bumetanide in Urine at Levels Required for
Doping Analysis. Microchim. Acta 2017, 184, 4117–4124. DOI:
10.1007/s00604-017-2443-5.
[63] Narapusetti, A.; Sundar Bethanabhatla, S.; Sockalingam, A.;
Rao Pilli, N. LC–MS/MS Assay for Acetazolamide, a Carbonic
Anhydrase Inhibitor in Human Plasma and Its Clinical
Application. J. Young Pharm. 2015, 7, 438–445. DOI: 10.5530/
jyp.2015.4s.5.
[64] Dubey, R.; Ghosh, M. Simultaneous Determination and
Pharmacokinetic Study of Losartan, Losartan Carboxylic Acid,
Ramipril, Ramiprilat, and Hydrochlorothiazide in Rat Plasma
by a Liquid Chromatography/Tandem Mass Spectrometry
Method. Sci. Pharm. 2015, 83, 107–124. DOI: 10.3797/sci-
pharm.1410-15.
[65] Gong, C. B.; Wei, Y. B.; Liu, L. T.; Zheng, A. X.; Yang, Y. H.;
Chow, C. F.; Tang, Q. Photoresponsive Hollow Molecularly
Imprinted Polymer for Trace Triamterene in Biological
Samples. Mater. Sci. Eng. C Mater. Biol. Appl. 2017, 76, 568–
578. DOI: 10.1016/j.msec.2017.03.135.
[66] Farnoudian-Habibi, A.; Kangari, S.; Massoumi, B.; Jaymand,
M. Determination of Losartan Potassium in the Presence of
Hydrochlorothiazide via a Combination of Magnetic Solid
Phase Extraction and Fluorometry Techniques in Urine
Samples. RSC Adv. 2015, 5, 102895–102903. DOI: 10.1039/
C5RA20117A.
[67] Bradshaw, D.; Garai, A.; Huo, J. Metal–Organic Framework
Growth at Functional Interfaces: thin Films and Composites
for Diverse Applications. Chem. Soc. Rev. 2012, 41, 2344–
2381. DOI: 10.1039/c1cs15276a.
[68] Seidi, S.; Tajik, M.; Baharfar, M.; Rezazadeh, M. Micro Solid-
Phase Extraction (Pipette Tip and Spin Column) and Thin
Film Solid-Phase Microextraction: Miniaturized Concepts for
Chromatographic Analysis. TrAC, Trends Anal. Chem. 2019,
118, 810–827. DOI: 10.1016/j.trac.2019.06.036.
[69] Scigalski, P.; Kosobucki, P. Recent Materials Developed for
Dispersive Solid Phase Extraction. Molecules 2020, 25, 4869.
DOI: 10.3390/molecules25214869.
[70] Buszewski, B.; Szultka, M. Past, Present, and Future of Solid
Phase Extraction: A Review. Crit. Rev. Anal. Chem. 2012, 42,
198–213. DOI: 10.1080/07373937.2011.645413.
[71] Fahad Alajmi, M.; Hussain, A.; Noeman Taqui, S.; Ali, I. Solid
Phase Micro Membrane Tip Extraction and Capillary
Electrophoresis of Metformin in Urine Samples. CPA 2017,
13, 403. DOI: 10.2174/1573412912666160804124750.
[72] Huang, S.; Chen, G.; Ye, N.; Kou, X.; Zhu, F.; Shen, J.;
Ouyang, G. Solid-Phase Microextraction: An Appealing
Alternative for the Determination of Endogenous substances –
A Review. Anal. Chim. Acta. 2019, 1077, 67–86. DOI: 10.1016/
j.aca.2019.05.054.
30 Y.-J. LIU ET AL.
[73] Yu, M.-Y.; Yang, X.-Q.; Fan, R.; Zheng, Y.-K.; Shi, J.-B.;
Zheng, Q. Non-Target Screening Analysis of Volatile Organic
Compounds in Drinking Water by Headspace-Solid Phase
Microextraction Gas Chromatography-Mass Spectrometry.
Chin. J. Anal. Chem. 2020, 48, 1228–1235. DOI: 10.1016/
S1872-2040(20)60044-5.
[74] Sobczak, Ł.; Kołodziej, D.; Gory nski, K. Modifying Current
Thin-Film Microextraction (TFME) Solutions for Analyzing
Prohibited Substances: Evaluating New Coatings Using Liquid
Chromatography. J. Pharm. Anal. 2022, 12, 470–480. DOI: 10.
1016/j.jpha.2021.12.007.
[75] Reyes-Garces, N.; Bojko, B.; Pawliszyn, J. High Throughput
Quantification of Prohibited Substances in Plasma Using Thin
Film Solid Phase Microextraction. J. Chromatogr. A 2014,
1374, 40–49. DOI: 10.1016/j.chroma.2014.11.047.
[76] Al-Hadithi, N.; Saad, B.; Grote, M. A Solid Bar
Microextraction Method for the Liquid Chromatographic
Determination of Trace Diclofenac, Ibuprofen and
Carbamazepine in River Water. Microchim. Acta 2011, 172,
31–37. DOI: 10.1007/s00604-010-0463-5.
[77] Neng, N.; Silva, A.; Nogueira, J. Adsorptive Micro-Extraction
Techniques—Novel Analytical Tools for Trace Levels of Polar
Solutes in Aqueous Media. J. Chromatogr. A 2010, 1217,
7303–7310. DOI: 10.1016/j.chroma.2010.09.048.
[78] Ide, A. H.; Nogueira, J. M. F. New-Generation Bar Adsorptive
Microextraction (BAmuE) Devices for a Better Eco-User-
Friendly Analytical approach-Application for the
Determination of Antidepressant Pharmaceuticals in Biological
Fluids. J. Pharm. Biomed. Anal. 2018, 153, 126–134. DOI: 10.
1016/j.jpba.2018.02.001.
[79] Anastassiades, M.; Lehotay, S. J.; Stajnbaher, D.; Schenck, F. J.
Fast and Easy Multiresidue Method Employing Acetonitrile
Extraction/Partitioning and “Dispersive Solid-Phase
Extraction” for the Determination of Pesticide Residues in
Produce. J. AOAC Int. 2003, 86, 412–431. DOI: 10.1093/jaoac/
86.2.412.
[80] Rahman, N.; Sameen, S.; Kashif, M. Application of Box-
Behnken Design and Desirability Function in the
Optimization of Spectrophotometric Method for the
Quantification of WADA Banned Drug: Acetazolamide. J.
Mol. Liq. 2019, 274, 270–277. DOI: 10.1016/j.molliq.2018.10.
120.
[81] Dangre, P.; Sawale, V.; Meshram, S.; Gunde, M. Development
and Validation of RP-HPLC Method for the Simultaneous
Estimation of Eprosartan Mesylate and Chlorthalidone in
Tablet Dosage Form. Int. J. PharmTech Res. 2015, 8, 163.
[82] Hadjikinova, R.; Petkova, N.; Hadjikinov, D.; Denev, P.;
Hrusavov, D. Development and Validation of HPLC-RID
Method for Determination of Sugars and Polyols. J.
Pharmaceut. Sci. Res. 2017, 9, 1263.
[83] El-Bagary, R. I.; Elkady, E. F.; Mowaka, S.; Attallah, M. A. A
Validated HPLC Method for Simultaneous Determination of
Perindopril Arginine, Amlodipine, and Indapamide:
Application in Bulk and in Different Pharmaceutical Dosage
Forms. J. AOAC Int. 2017, 100, 992–999. DOI: 10.5740/jaoa-
cint.16-0279.
[84] Naguib, I. A.; Abdelaleem, E. A.; Emam, A. A.; Ali, N. W.;
Abdallah, F. F. Development and Validation of HPTLC and
Green HPLC Methods for Determination of Furosemide,
Spironolactone and Canrenone, in Pure Forms, Tablets and
Spiked Human Plasma. Biomed. Chromatogr. 2018, 32, e4304.
DOI: 10.1002/bmc.4304.
[85] Patel, A. S.; Sayyed, S. N.; Lajporiya, I. M.; Manjra, U. M.;
Ahmed, A.; Khan, G. J. An Eco-Friendly RP-HPLC and UV-
Method Development and Validation for an Estimation of
Tolvaptan in Bulk and Tablet Dosage Form Followed by
Forced Degradation Studies. JPRI. 2021, 33(42B), 271–286.
DOI: 10.9734/jpri/2021/v33i42B32446.
[86] Chen, F.; Fang, B.; Li, P.; Wang, S. Simultaneous
Determination of Five Diuretic Drugs Using Quantitative
Analysis of Multiple Components by a Single Marker. BMC
Chem. 2021, 15, 39.
[87] Abd El-Hay, S. S.; Hashem, H.; Gouda, A. A. High
Performance Liquid Chromatography for Simultaneous
Determination of Xipamide, Triamterene and
Hydrochlorothiazide in Bulk Drug Samples and Dosage
Forms. Acta Pharm. 2016, 66, 109–118. DOI: 10.1515/acph-
2016-0022.
[88] Anderson, J. F. F.; Gerlin, M. C. G.; Sversut, R. A.; Oliveira,
L. C. S.; Singh, A. K.; Amaral, M. S.; Kassab, N. M.
Development and Validation of an Isocratic HPLC Method for
Simultaneous Determination of Quaternary Mixtures of
Antihypertensive Drugs in Pharmaceutical Formulations. Acta
Chromatogr. 2017, 29, 95–110. DOI: 10.1556/1326.2017.29.1.9.
[89] Manchanda, S.; Sahoo, P.; Majumdar, D. RP-HPLC Method
Development and Validation for the Estimation of
Acetazolamide in Bulk Drug and Formulations with Forced
Degradation Studies. Pharm. Lett. 2016, 8, 338.
[90] _
Stolarczyk, M.; Hubicka, U.; Zuromska-Witek, B.; Krzek, J.
Simultaneous Determination of Eight Hypotensive Drugs of
Various Chemical Groups in Pharmaceutical Preparations by
HPLC-DAD. J. AOAC Int. 2015, 98, 1542–1548. DOI: 10.
5740/jaoacint.15-022.
[91] Chinta, S. R.; Paidikondala, K.; Katari, N. K.; Dongala, T.;
Ediga, S. G.; Marisetti, V. M. Reverse-Phase LC Method
Development and Validation for the Quantification of
Acetazolamide and Its Specified and Unspecified Degradation
Products in Hard Gelatin Capsule Formulations. J. Iran.
Chem. Soc. 2022, 19, 775–784. DOI: 10.1007/s13738-021-
02341-6.
[92] Aa, M.; Kannappan, N.; Cb, M. K. Analytical Method
Development and Validation for the Determination of
Hydrochlorothiazide, Amlodipine Besylate and Telmisartan
Hydrochloride in Multicomponent Tablet Dosage Form and in
Biorelevant Media (FaSSIF) by RP-HPLC techniques. 2015.
[93] Hammouda, M. E.; Abu El-Enin, M. A.; El-Sherbiny, D. T.;
El-Wasseef, D. R.; El-Ashry, S. M. Simultaneous
Determination of Enalapril and Hydrochlorothiazide in
Pharmaceutical Preparations Using Microemulsion Liquid
Chromatography. J. Chromatogr. Sci. 2015, 53, 90–96. DOI:
10.1093/chromsci/bmu024.
[94] Dong, Y.; Yan, K.; Ma, Y.; Yang, Z.; Zhao, J.; Ding, J. A
Modified LC-MS/MS Method to Simultaneously Quantify
Glycerol and Mannitol Concentrations in Human Urine for
Doping Control Purposes. J. Chromatogr. B Analyt. Technol.
Biomed. Life Sci. 2016, 1022, 153–158. DOI: 10.1016/j.jchromb.
2016.04.023.
[95] Helmlin, H. J.; Murner, A.; Steiner, S.; Kamber, M.; Weber, C.;
Geyer, H.; Guddat, S.; Schanzer, W.; Thevis, M. Detection of
the Diuretic Hydrochlorothiazide in a Doping Control Urine
Sample as the Result of a Non-Steroidal anti-Inflammatory
Drug (NSAID) Tablet Contamination. Forensic Sci. Int. 2016,
267, 166–172. DOI: 10.1016/j.forsciint.2016.08.029.
[96] Avataneo, V.; De Nicolo, A.; Rabbia, F.; Sciandra, M.; Tosello,
F.; Cusato, J.; Perlo, E.; Fatiguso, G.; Allegra, S.; Favata, F.;
et al. A Simple UHPLC-PDA Method with a Fast Dilute-and-
Shot Sample Preparation for the Quantification of Canrenone
and Its Prodrug Spironolactone in Human Urine Samples. J.
Pharmacol. Toxicol. Methods. 2018, 94, 29–35. DOI: 10.1016/j.
vascn.2018.08.003.
[97] Gheddar, L.; Raul, J. S.; Kintz, P. First Identification of a
Diuretic, Hydrochlorothiazide, in Hair: Application to a
Doping Case and Interpretation of the Results. Drug Test.
Anal. 2019, 11, 157–161. DOI: 10.1002/dta.2445.
[98] Dahmana, N.; Gabriel, D.; Gurny, R.; Kalia, Y. N.
Development and Validation of a Fast and Sensitive UHPLC-
ESI-MS Method for the Simultaneous Quantification of
Spironolactone and Its Metabolites in Ocular Tissues. Biomed.
Chromatogr. 2018, 32, e4287. DOI: 10.1002/bmc.4287.

[99] Richter, L. H. J.; Jacobs, C. M.; Mahfoud, F.; Kindermann, I.;
Bohm, M.; Meyer, M. R. Development and Application of a
LC-HRMS/MS Method for Analyzing Antihypertensive Drugs
in Oral Fluid for Monitoring Drug Adherence. Anal. Chim.
Acta. 2019, 1070, 69–79. DOI: 10.1016/j.aca.2019.04.026.
[100] Chen, F.; Fang, B.; Wang, S. A Fast and Validated HPLC
Method for Simultaneous Determination of Dopamine,
Dobutamine, Phentolamine, Furosemide, and Aminophylline
in Infusion Samples and Injection Formulations. J. Anal.
Methods Chem. 2021, 2021,):8821126. DOI: 10.1155/2021/
8821126.
[101] Lee, J.-H.; An, T.-G.; Kim, S. J.; Shim, W.-S.; Lee, K.-T.
Development of Liquid Chromatography Tandem Mass
Spectrometry Method for Determination of Spironolactone in
Human Plasma: application to a Bioequivalence Study of
Daewon Spiracton TabletV R (Spironolactone 50 mg). J.
Pharmaceut. Invest. 2015, 45, 601–609. DOI: 10.1007/s40005-
015-0197-9.
[102] Alrabiah, H.; Kadi, A. A.; Attwa, M. W.; Mostafa, G. A. E.
Development and Validation of an HPLC-MS/MS Method for
the Determination of Arginine-Vasopressin Receptor Blocker
Conivaptan in Human Plasma and Rat Liver Microsomes:
application to a Metabolic Stability Study. Chem. Cent. J. 2018,
12, 47.
[103] Johannsen, J. O.; Reuter, H.; Hoffmann, F.; Blaich, C.; Wiesen,
M. H. J.; Streichert, T.; Muller, C. Reliable and Easy-to-Use
LC-MS/MS-Method for Simultaneous Determination of the
Antihypertensives Metoprolol, Amlodipine, Canrenone and
Hydrochlorothiazide in Patients with Therapy-Refractory
Arterial Hypertension. J. Pharm. Biomed. Anal. 2019, 164,
373–381. DOI: 10.1016/j.jpba.2018.11.002.
[104] Ebeid, W. M.; Elkady, E. F.; El-Zaher, A. A.; El-Bagary, R. I.;
Patonay, G. Simultaneous Determination of Aliskiren
Hemifumarate, Amlodipine Besylate and Hydrochlorothiazide
in Spiked Human Plasma Using UPLC-MS/MS. J. Chromatogr.
Sci. 2015, 53, 1178–1184. DOI: 10.1093/chromsci/bmu213.
[105] Aydoǧmuş, Z. Simultaneous Determination of Aliskiren,
Amlodipine and Hydrochlorothiazide in Spiked Human
Plasma and Urine by High Performance Liquid
Chromatography. J. Anal. Chem. 2015, 70, 502–509. DOI: 10.
1134/S1061934815040176.
[106] Nahar, L.; Onder, A.; Sarker, S. D. A Review on the Recent
Advances in HPLC, UHPLC and UPLC Analyses of Naturally
Occurring Cannabinoids (2010-2019). Phytochem. Anal. 2020,
31, 413–457. DOI: 10.1002/pca.2906.
[107] Hemdan, A.; Magdy, R.; Farouk, M. Response Surface Design
as a Powerful Tool for the Development of Environmentally
Benign HPLC Methods for the Determination of Two
Antihypertensive Combinations: Greenness Assessment by
Two Green Analytical Chemistry Evaluation Tools. J. Sep. Sci.
2018, 41, 3213–3223. DOI: 10.1002/jssc.201800317.
[108] Gillium, C.; Friciu, M.; Abatzoglou, N.; Leclair, G. Validation
of a Stability-Indicating HPLC-UV Method for the
Quantification of Acetazolamide in Oral-Mix and Oral-Mix
SF. MethodsX 2020, 7, 100844. DOI: 10.1016/j.mex.2020.
100844.
[109] Naguib, I. A.; Abdelaleem, E. A.; Draz, M. E.; Zaazaa, H. E.
Development and Validation of RP-HPLC Method for
Determination of Hydrochlorothiazide, Amiloride
Hydrochloride and Related Impurities in Bulk and
Pharmaceutical Dosage Forms. Anal. Chem. Lett. 2015, 5, 85–
93. DOI: 10.1080/22297928.2015.1026394.
[110] Patil, M. P. N. HPLC Method Development–A Review. J.
Pharmaceut. Res. Educ. 2017, 1, 243.
[111] Tolba, M. M.; Belal, F. Two Liquid Chromatographic
Approaches for the Simultaneous Determination of Xipamide
and Its Degradation Product (2,6-Xylidine) Using Time-
Programmed Fluorescence Detection. Luminescence 2017, 32,
491–501. DOI: 10.1002/bio.3203.
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 31
[112] Shaaban, H. New Insights into Liquid Chromatography for
More Eco-Friendly Analysis of Pharmaceuticals. Anal. Bioanal.
Chem. 2016, 408, 6929–6944. DOI: 10.1007/s00216-016-9726-
2.
[113] Peris-Garcıa, E.; Pankajkumar-Patel, N.; Ruiz-Angel, M. J.;
Carda-Broch, S.; Garcıa-Alvarez-Coque, M. C. Oil-In-Water
Microemulsion Liquid Chromatography. Separation &
Purification Reviews 2020, 49, 89–111. DOI: 10.1080/15422119.
2018.1524386.
[114] Pankajkumar-Patel, N.; Peris-Garcia, E.; Ruiz-Angel, M. J.;
Carda-Broch, S.; Garcia-Alvarez-Coque, M. C. Modulation of
Retention and Selectivity in Oil-in-Water Microemulsion
Liquid Chromatography: A Review. J. Chromatogr. A 2019,
1592, 91–100. DOI: 10.1016/j.chroma.2019.01.046.
[115] Li, L.; Lai, C.; Xuan, X.; Gao, C.; Li, N. Simultaneous
Determination of Hydrochlorothiazide and Losartan
Potassium in Osmotic Pump Tablets by Microemulsion Liquid
Chromatography. J. Chromatogr. Sci. 2016, 54, 1415–1420.
DOI: 10.1093/chromsci/bmw101.
[116] Carlucci, G.; Palumbo, G.; Mazzeo, P.; Quaglia, M. G.
Simultaneous Determination of Losartan and
Hydrochlorothiazide in Tablets by High-Performance Liquid
Chromatography. J. Pharm. Biomed. Anal. 2000, 23, 185–189.
DOI: 10.1016/s0731-7085(00)00268-5.
[117] Awad, H.; Khamis, M. M.; El-Aneed, A. Mass Spectrometry,
Review of the Basics: Ionization. Appl. Spectrosc. Rev. 2015,
50, 158–175. DOI: 10.1080/05704928.2014.954046.
[118] El-Aneed, A.; Cohen, A.; Banoub, J. Mass Spectrometry,
Review of the Basics: Electrospray, MALDI, and Commonly
Used Mass Analyzers. Appl. Spectrosc. Rev. 2009, 44, 210–230.
DOI: 10.1080/05704920902717872.
[119] Holcapek, M.; Jirasko, R.; Lisa, M. Recent Developments in
Liquid Chromatography-Mass Spectrometry and Related
Techniques. J. Chromatogr. A 2012, 1259, 3–15. DOI: 10.1016/
j.chroma.2012.08.072.
[120] Lo Faro, A. F.; Tini, A.; Gottardi, M.; Pirani, F.; Sirignano, A.;
Giorgetti, R.; Busardo, F. P. Development and Validation of a
Fast Ultra-High-Performance Liquid Chromatography Tandem
Mass Spectrometry Method for Determining Carbonic
Anhydrase Inhibitors and Their Metabolites in Urine and
Hair. Drug Test. Anal. 2021, 13, 1552–1560. DOI: 10.1002/dta.
3055.
[121] Ki, N.-Y.; Hur, J.; Kim, B. H.; Kim, K. H.; Moon, B. J.; Oh,
H. B.; Hong, J. Rapid Screening of Sulfonamides in Dietary
Supplements Based on Extracted Common Ion Chromatogram
and Neutral Loss Scan by LC-Q/TOF-Mass Spectrometry. J.
Food Drug Anal. 2019, 27, 164–174. DOI: 10.1016/j.jfda.2018.
08.006.
[122] Eliuk, S.; Makarov, A. Evolution of Orbitrap Mass
Spectrometry Instrumentation. Annu. Rev. Anal. Chem. (Palo
Alto Calif.) 2015, 8, 61–80. DOI: 10.1146/annurev-anchem-
071114-040325.
[123] Abushareeda, W.; Vonaparti, A.; Saad, K. A.; Almansoori, M.;
Meloug, M.; Saleh, A.; Aguilera, R.; Angelis, Y.; Horvatovich,
P. L.; Lommen, A.; et al. High Resolution Full Scan Liquid
Chromatography Mass Spectrometry Comprehensive
Screening in Sports Antidoping Urine Analysis. J. Pharm.
Biomed. Anal. 2018, 151, 10–24. DOI: 10.1016/j.jpba.2017.12.
025.
[124] Svan, A.; Hedeland, M.; Arvidsson, T.; Pettersson, C. E. The
Differences in Matrix Effect between Supercritical Fluid
Chromatography and Reversed Phase Liquid Chromatography
Coupled to ESI/MS. Anal. Chim. Acta. 2018, 1000, 163–171.
DOI: 10.1016/j.aca.2017.10.014.
[125] Siddiqui, M. R.; AlOthman, Z. A.; Rahman, N. Analytical
Techniques in Pharmaceutical Analysis: A Review. Arab. J.
Chem. 2017, 10, S1409–S1421. DOI: 10.1016/j.arabjc.2013.04.
016.
[126] Subramanian, V.; Nagappan, K.; Sandeep Mannemala, S.
Optimization and Validation of a Sensitive Method for HPLC-
32 Y.-J. LIU ET AL.

PDA Simultaneous Determination of Torasemide and
Spironolactone in Human Plasma Using Central Composite
Design. Acta Chim. Slov. 2015, 62, 633–641. DOI: 10.17344/
acsi.2014.1262.
[127] Antal, I.; Koneracka, M.; Zavisova, V.; Kubovcikova, M.;
Kormosh, Z.; Kopcansky, P. Statins Determination: A Review
of Electrochemical Techniques. Crit. Rev. Anal. Chem. 2017,
47, 474–489. DOI: 10.1080/10408347.2017.1332973.
[128] Mansano, G. R.; Pires Eisele, A. P.; Sartori, E. R.
Electrochemical Evaluation of a Boron-Doped Diamond
Electrode for Simultaneous Determination of an
Antihypertensive Ternary Mixture of Amlodipine,
Hydrochlorothiazide and Valsartan in Pharmaceuticals. Anal.
Methods 2015, 7, 1053–1060. DOI: 10.1039/C4AY02511C.
[129] Bulut, _
I. Simultaneous Square-Wave Voltammetric
Determination of Acetazolamide and Theophylline in
Pharmaceutical Formulations. Russ. J. Electrochem. 2016, 52,
427–434. DOI: 10.1134/S1023193516050025.
[130] Karimi, R.; Gholivand, M. B.; Amiri, M. Monitoring of
Triamterene and Hydrochlorothiazide at Carbonic Materials
Modified Electrode. Electroanal. Chem. 2019, 847, 113176.
DOI: 10.1016/j.jelechem.2019.05.058.
[131] Machini, W. B.; David-Parra, D. N.; Teixeira, M. F.
Electrochemical Investigation of the Voltammetric
Determination of Hydrochlorothiazide Using a Nickel
Hydroxide Modified Nickel Electrode. Mater. Sci. Eng. C
Mater. Biol. Appl. 2015, 57, 344–348. DOI: 10.1016/j.msec.
2015.07.035.
[132] Said, M. I.; Rageh, A. H.; F. A. M., Abdel-Aal. Fabrication of
Novel Electrochemical Sensors Based on Modification with
Different Polymorphs of MnO2 Nanoparticles. Application to
Furosemide Analysis in Pharmaceutical and Urine Samples.
RSC Adv. 2018, 8, 18698–18713. DOI: 10.1039/c8ra02978d.
[133] Kor, K.; Zarei, K. Development and Characterization of an
Electrochemical Sensor for Furosemide Detection Based on
Electropolymerized Molecularly Imprinted Polymer. Talanta
2016, 146, 181–187. DOI: 10.1016/j.talanta.2015.08.042.
[134] Medeiros, R. A.; Baccarin, M.; Fatibello-Filho, O.; Rocha-Filho,
R. C.; Deslouis, C.; Debiemme-Chouvy, C. Comparative Study
of Basal-Plane Pyrolytic Graphite, Boron-Doped Diamond,
and Amorphous Carbon Nitride Electrodes for the
Voltammetric Determination of Furosemide in Pharmaceutical
and Urine Samples. Electrochim. Acta 2016, 197, 179–185.
DOI: 10.1016/j.electacta.2015.10.065.
[135] Kumar, N.; Goyal, R. N. Melamine/Fe3O4 Nanoparticles Based
Molecular Imprinted Highly Sensitive Sensor for
Determination of Hydrochlorothiazide: An Antihypertensive
Drug. J. Electrochem. Soc. 2017, 164, B240–B246. DOI: 10.
1149/2.1451706jes.
[136] Khorshed, A. A.; Khairy, M.; Banks, C. E. Electrochemical
Determination of Antihypertensive Drugs by Employing
Costless and Portable Unmodified Screen-Printed Electrodes.
Talanta 2019, 198, 447–456. DOI: 10.1016/j.talanta.2019.01.
117.
[137] Wang, Y.; Cheng, J.; Liu, X.; Ding, F.; Zou, P.; Wang, X.;
Zhao, Q.; Rao, H. C3N4 Nanosheets/Metal–Organic
Framework Wrapped with Molecularly Imprinted Polymer
Sensor: Fabrication, Characterization, and Electrochemical
Detection of Furosemide. ACS Sustainable Chem. Eng. 2018, 6,
16847–16858. DOI: 10.1021/acssuschemeng.8b04179.
[138] Esfandiari Baghbamidi, S. Voltammetric Sensor Based on 1-
Benzyl-4-Ferrocenyl-1H- [1,2,3]-Triazole/Carbon Nanotube
Modified Glassy Carbon Electrode; Detection of
Hydrochlorothiazide in the Presence of Propranolol. Int. J.
Electrochem. Sci. 2016, 11(12), 10874–10883. DOI: 10.20964/
2016.12.92.
[139] Calegari, F.; Roberto de Oliveira, P.; Marcolino Junior, L. H.;
Bergamini, M. F. A Carbon Black Composite Electrode for
Flow Injection Amperometric Determination of
Hydrochlorothiazide. Anal. Methods 2019, 11, 2422–2427.
DOI: 10.1039/C9AY00555B.
[140] Mundaca-Uribe, R.; Diego, M. d.; Henrıquez-Aedo, K.;
Aranda, M.; Pe~ na-Farfal, C. Development and Characterization
of a Sensor Based on Carbon Nanofibers: Application to
Acetazolamide Determination in Pharmaceuticals and
Biological Fluids. J. Chil. Chem. Soc. 2019, 64, 4382–4385.
DOI: 10.4067/s0717-97072019000104382.
[141] Tian, F.; Li, H.; Li, M.; Li, C.; Lei, Y.; Yang, B. A Tantalum
Electrode Coated with Graphene Nanowalls for Simultaneous
Voltammetric Determination of Dopamine, Uric Acid, L-
Tyrosine, and Hydrochlorothiazide. Microchim. Acta 2017,
184, 1611–1619. DOI: 10.1007/s00604-017-2154-y.
[142] Tantawy, M. A.; El Fiky, H. A.; Badawey, A. M.; Abd El
Ghany, M. F.; Fares, N. V. A Novel Glassy Carbon Electrode
Modified with Multi-Walled Carbon Nanotubes for
Potentiometric Xipamide Determination. J. Electrochem. Soc.
2021, 168, 056506. DOI: 10.1149/1945-7111/abfcdb.
[143] Lourencao, B. C.; Medeiros, R. A.; Fatibello-Filho, O.
Simultaneous Determination of Antihypertensive Drugs by
Flow Injection Analysis Using Multiple Pulse Amperometric
Detection with a Cathodically Pretreated Boron-Doped
Diamond Electrode. Electroanal. Chem. 2015, 754, 154–159.
DOI: 10.1016/j.jelechem.2015.06.022.
[144] Pereira, P. F.; da Silva, W. P.; Marra, M. C.; Mu~noz, R. A. A.;
Richter, E. M. A High-Throughput BIA-MPA Method for the
Simultaneous Determination of Amiloride and Furosemide.
Anal. Methods 2016, 8, 7959–7965. DOI: 10.1039/
C6AY02506D.
[145] Silva, E. F.; Tanaka, A. A.; Fernandes, R. N.; Munoz, R. A. A.;
da Silva, I. S. Batch Injection Analysis with Electrochemical
Detection for the Simultaneous Determination of the Diuretics
Furosemide and Hydrochlorothiazide in Synthetic Urine and
Pharmaceutical Samples. Microchem. J. 2020, 157, 105027.
DOI: 10.1016/j.microc.2020.105027.
[146] Coelho, J. H.; Eisele, A. P. P.; Valezi, C. F.; Mattos, G. J.;
Schirmann, J. G.; Dekker, R. F. H.; Barbosa-Dekker, A. M.;
Sartori, E. R. Exploring the Exocellular Fungal Biopolymer
Botryosphaeran for Laccase-Biosensor Architecture and
Application to Determine Dopamine and Spironolactone.
Talanta 2019, 204, 475–483. DOI: 10.1016/j.talanta.2019.06.
033.
[147] Machini, W. B.; Teixeira, M. F. Analytical Development of a
Binuclear Oxo-Manganese Complex Bio-Inspired on Oxidase
Enzyme for Doping Control Analysis of Acetazolamide.
Biosens. Bioelectron. 2016, 79, 442–448. DOI: 10.1016/j.bios.
2015.12.026.
[148] Saini, A.; Kaur, N.; Singh, N. A Highly Fluorescent Sensor
Based on Hybrid Nanoparticles for Selective Determination of
Furosemide in Aqueous Medium. Sens. Actuators, B 2016, 228,
221–230. DOI: 10.1016/j.snb.2016.01.026.
[149] Hudari, F. F.; Souza, J. C.; Zanoni, M. V. B. Adsorptive
Stripping Voltammetry for Simultaneous Determination of
Hydrochlorothiazide and Triamterene in Hemodialysis
Samples Using a Multi-Walled Carbon Nanotube-Modified
Glassy Carbon Electrode. Talanta 2018, 179, 652–657. DOI:
10.1016/j.talanta.2017.11.071.
[150] Ksenofontov, A. A.; Lukanov, M. M.; Antina, E. V.
Fluorescent Detection of Loop Diuretics by Sensors Based on
Zinc(II) Bis(Dipyrromethenate)s. Dyes Pigm. 2020, 179,
108389. DOI: 10.1016/j.dyepig.2020.108389.
[151] Munyendo, L.; Njoroge, D.; Hitzmann, B. The Potential of
Spectroscopic Techniques in Coffee Analysis—A Review.
Processes 2021, 10, 71. DOI: 10.3390/pr10010071.
[152] Binh, T. T.; Phuong Tram, L. T.; Van Hop, N.; Giang Chau,
N. D.; Luu, N. D.; Quynh Trang, N. T. Simultaneous
Determination of Hydrochlorothiazide and Losartan
Potassium in Pharmaceutical Product by UV-Vis
Spectrophotometric Method with Kalman Filter Algorithm. J.

Anal. Methods Chem. 2021, 2021:2754133. DOI: 10.1155/2021/
2754133.
[153] Chandarana, C. V.; Prajapati, P. R.; Jani, G. K. Quantification
of Spironolactone by Fourier Transform Infrared
Spectrophotometry in Bulk and Tablet Dosageform. Vib.
Spectrosc. 2019, 100, 185–190. DOI: 10.1016/j.vibspec.2018.12.
006.
[154] Sanchez, F. G.; Diaz, A. N.; Lopez Guerrero, M. M. Time-
Resolved Spectroscopy for Selective Determination of
Fluorescent Diuretics. Spectrosc. Lett. 2015, 48, 481–486. DOI:
10.1080/00387010.2014.895385.
[155] Mohammed, N. M. S.; Abdo, H. R.; Hassan, H. M. Method
Development and Validation of Simultaneous Determination
of Hydrochlorothiazide And Losartan In Tablet Dosage Form
by RP-HPLC. International journal of Pharmaceutical sciences
and research, 2019, 10(1), 227–231. DOI:10.13040/IJPSR.0975-
8232.10(1).1000-05.
[156] McGown, L. B.; Bright, F. V. Phase-Resolved Fluorescence
Spectroscopy. Anal. Chem. 1984, 56, 1400A–1417A. DOI: 10.
1021/ac00277a001.
[157] Li, L.; Wang, Y.; Zhang, W.; Yu, S.; Wang, X.; Gao, N. New
Advances in Fluorescence Excitation-Emission Matrix
Spectroscopy for the Characterization of Dissolved Organic
Matter in Drinking Water Treatment: A Review. Chem. Eng. J.
2020, 381, 122676. DOI: 10.1016/j.cej.2019.122676.
[158] Hu, Y.; Wu, H. L.; Yin, X. L.; Gu, H. W.; Kang, C.; Xiang,
S. X.; Xia, H.; Yu, R. Q. Chemometrics-Assisted
Determination of Amiloride and Triamterene in Biological
Fluids with Overlapped Peaks and Unknown Interferences.
Bioanalysis 2015, 7, 1685–1697. DOI: 10.4155/bio.15.88.
[159] Albertsdottir, A. D.; Van Gansbeke, W.; Van Eenoo, P.; Polet,
M. Enabling the Inclusion of Non-Hydrolysed sulfated long
term Anabolic Steroid Metabolites in a Screening for Doping
Substances by Means of Gas Chromatography Quadrupole
Time-of-Flight Mass Spectrometry. J. Chromatogr. A 2021,
1642, 462039. DOI: 10.1016/j.chroma.2021.462039.
[160] Polet, M.; Van Gansbeke, W.; Van Eenoo, P. Development
and Validation of an Open Screening Method for Doping
Substances in Urine by Gas Chromatography Quadrupole
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 33
Time-of-Flight Mass Spectrometry. Anal. Chim. Acta. 2018,
1042, 52–59. DOI: 10.1016/j.aca.2018.08.050.
[161] Begou, O.; Drabert, K.; Theodoridis, G.; Tsikas, D. GC-NICI-
MS Analysis of Acetazolamide and Other Sulfonamide (R-
SO2-NH2) Drugs as Pentafluorobenzyl Derivatives [R-SO2-
N(PFB)2] and Quantification of Pharmacological
Acetazolamide in Human Urine. J. Pharm. Anal. 2020, 10, 49–
59. DOI: 10.1016/j.jpha.2019.11.006.
[162] Grapp, M.; Kaufmann, C.; Streit, F.; Binder, L. Systematic
Forensic Toxicological Analysis by Liquid-Chromatography-
Quadrupole-Time-of-Flight Mass Spectrometry in Serum and
Comparison to Gas Chromatography-Mass Spectrometry.
Forensic Sci. Int. 2018, 287, 63–73. DOI: 10.1016/j.forsciint.
2018.03.039.
[163] Wang, L.; Du, Z.; Guan, Y.; Wang, B.; Pei, Y.; Zhang, L.;
Fang, M. Identifying Absorbable Bioactive Constituents of
Yupingfeng Powder Acting on COVID-19 through Integration
of UPLC-Q/TOF-MS and Network Pharmacology Analysis.
Chin. Herb. Med. 2022, 14, 283–293. DOI: 10.1016/j.chmed.
2022.02.001.
[164] Dolowy, M. Influence of Different Chromatographic
Conditions on the level of Detection and Quantitation of
Spironolactone by Means of TLC-Densitometry. J. Anal.
Methods Chem. 2019, 2019, 8792783.
[165] Ramu, B.; Chittela, K. B. High Performance Thin Layer
Chromatography and Its Role Pharmaceutical Industry. Open
Sci. J. Biosci. Bioeng. 2018, 5, 29–34.
[166] Li, L.; Huang, Y.; Zhao, W.; Zhang, G.; Zhang, H.; Chen, A.
Simultaneous Separation and Rapid Determination of
Spironolactone and Its Metabolite Canrenone in Different
Pharmaceutical Formulations and Urinary Matrices by
Capillary Zone Electrophoresis. J. Sep. Sci. 2016, 39, 2869–
2875. DOI: 10.1002/jssc.201600255.
[167] Fayez, Y. M.; Hegazy, M. A. Micellar Electrokinetic
Chromatography (MEKC) with Multiresponse Chemometric
Optimization for the Determination of Hydrochlorothiazide
and Coformulated Antihypertensives in the Presence of
Hydrochlorothiazide Major Impurity. J. Chromatogr. Sci. 2016,
54, 1050–1060. DOI: 10.1093/chromsci/bmw056.