Chapter 2

2.1 Introduction

Gemcitabine is a drug for treatment of multiple types of cancer, and a first-line anti-cancer drug that is used in
the treatment of pancreatic cancer. According to [2] in chapter 1, section 1.3, Gemcitabine is a prodrug that is
activated by phosphorylation before being absorbed by the body. When the drug enters the body cells, it can
either be deaminated to form dFdU in the plasma, kidney, or liver by cytidine deaminase, or it can be absorbed
in cancer cells by nucleoside transporters. Once in the cancer cells, the drug is phosphorylated by deoxycytidine
kinase to form active diphosphate and triphosphate nucleosides which are responsible for the drug’s cytotoxic
effects [5, 6]. Diphosphate leads to depleted pools of endogenous deoxyribonucleoside triphosphate through
inhibition of ribonucleoside reductase [7]. On the other hand, triphosphate induces cross-links when
incorporated into DNA, leading to inhibited cross-linked DNA repair [8]. Gemcitabine provides a novel
pyrimidine antimetabolite and deoxycytidine analog that exhibits anti-tumour characteristics by killing and
inhibiting DNA synthesis activity in cells [1, 2]. The chemical compounds known as nucleotides have two
different regions: a less polar ribose basic group [67], and a phosphate group (with a negative charge) which has
a pKa of approximately 1 [66]. On the other hand, the 2′-OH group of a nucleotide has a pKa of approximately
12.5. But with the presence of a phosphate group, this value may be modified in the range of 12.17-13.59 [68,
69]. As the number of phosphate group increases, so is the polarity (see figure 11). Unlike the high polarity of
diphosphates and triphosphates, the RP-HPLC column retains Gemcitabine [38, 39, 60, 65, and 70].

Figure 11: Increase in polarity as phosphate groups increase

One of the most common analytical techniques used in the analysis and quantification of Gemcitabine and its di
and triphosphate metabolites is HPLC. A number of chromatographic modes, such as IE-HPLC, IP-HPLC, and
RP-HPLC [2, 58, 96-99] have been employed to determine the nucleoside Gemcitabine monophosphate,
diphosphate, and triphosphate (dFdCTP). According to [98] an assay that is liquid chromatography-based and
with a weak anion exchange coupled to an MS/MS detection system was successfully produced. These three
components were separated by use of a pH gradient of the range 6-10.5 which is characterized by a lower
concentration of ammonium acetate in the HPLC mobile phase.

There are many methods that are documented for quantifying the amount of Gemcitabine in urine, plasma, and
body tissues by employing HPLC and a UV detector. HPLC has been developed to provide a rapid and sensitive
method for analysing clinical plasma samples [8-18]. Using HPLC-UV bioanalysis to quantify Gem in blood
plasma is very challenging due to the fact that Gemcitabine has a very short half-life as it is rapidly deaminated
[5, 6] to dFdU by cytidine deaminase. Furthermore, it is difficult to monitor low concentration levels of the
parent drug at subsequent sampling points since the sensitivity of ultraviolet is limited and cannot be easily
detected. For these reasons, chemical derivatization is often applied in varous categories of analytes for
enhancing chemical stability, ionization efficiency, as well as chromatographic separation. However, in most
cases, like in the case of Gemcitabine, the development of the HPLC method is hindered by low sample
recovery, weak sensitivity, analyte instability, and poor retention on HPLC.
HPLC analysis typically uses chemical derivatization with UV detection to improve selectivity or sensitivity.
Derivatization is a chemical reaction in which the prerequisites are the functional group from the compound of
interest, and the reaction group(s) of the derivatization reagent. This reaction changes the structure of analytes,
which changes their chemical and physical properties, HPLC retention, and extraction efficiency. For a polar
compound like Gemcitabine, poor sensitivity and poor retention on the HPLC column are the key challenges in
developing an efficient HPLC method. The derivatization process provides a lipophilic product and the
derivative can easily be extracted and concentrated during the sample preparation process. Additionally, there is
enhanced retention of the derivative on the chromatographic column, thus, reducing matrix effects as a result of
co-elution components [2, 3], and the libelled internal standards compensates for variation in reaction recovery
[6, 7 and 8].

The various functional groups in target compounds that react with derivatization reagents include: thiol, amine,
carbonyl, hydroxyl, and carboxyl [23-32]. Therefore, the classification of derivatization reagents into various
groups is based on how they react with different functional groups within the target compounds. For efficiency
and specificity, LC-MS analysis require an appropriate design and application of derivatization agents. There
are few studies that demonstrate the use of chromatography in separating amines and amino acids
simultaneously using derivatization and dansyl chloride [16]. DNS-Cl constitutes a sulfonyl group that can
undergo a dansylation-reaction with amines. DNS-Cl readily reacts with primary and secondary amino groups at
slightly alkaline pH to produce stable derivatives. The conditions for the derivatization reaction of Gemcitabine
vary from one research publication to another. However, the quantity of Gem and its metabolites derivatives
yielded is influenced by the reaction time, temperature, and pH of the medium. If one of these variables is
analysed, the cross-impact of all the variables would be neglected.

Figure 12: The structure of Gem and its metabolite derivatives (di and tri-phosphate).

Several bioanalytical methods have been developed to determine Gemcitabine. These include: HPLC with UV
detection, and UPLC with tandem mass spectroscopy for derivatization of Gem. The hydrophilic nature of Gem
hinders the achievement of high sensitivity as a result of poor ionization, and sometimes, due to weak retention
on columns. Kirstein et al. employed the HPLC-UV method to provide a LLQ of 500 ng/mL. On the other
hand, Wang et al. achieved a higher sensitivity of 80ng/mL using the HPLC-UV technique and ion-pairing
mobile phase by increasing Gem retention on the HPLC column, but was insufficient to accommodate
satisfactory pharmacokinetic profiles in the human body. Bowen et al. found that the LLQ of Gem using LC-
MS/MS was 20 ng/mL, although more efforts were needed to raise the retention time. In the current study, a
simple bioanalytical technique has been developed for extraction of cells, with increased sensitivity (LLOQ of
6.3 ng/mL) using Dns-Cl as the derivatization agent and RP-HPLC column, and analysis by LC-MS/MS and
HPLC-UV.
The challenge with this method is that Gem and its metabolites have different chemical structures; Gem
structure lack phosphate groups, making it hard to run all the three compounds in one sample. This implies that
to detect Gem metabolites using the HPLC method, there is need of an ion pair. HPLC-UV is only used to
determine Gem, but the relative high polarity of this compound reduces the resolution between the analyses on
RP-HPLC columns. Furthermore, the retention time and sensitivity of HPLC-UV of the analyses investigated
showed improvement using the derivatization method described. The table below shows

Table 4: Methods of HPLC published for quantification of DNS-GEM

Analytical Method Drugs Column Detecti Silent Features and Disadvantage

(Reference) on Advantages

HPLC-PDA Gem Zorber C-8 275nm Simple, Gradient elution

(Jansen Et Al, (5μm, 4.6 x 250 mm) sensitive
2000)
HPLC Gem ODS column 234nm Simple, accurate High organic
(Rao Et Al, 2007) (5μm, 4.6 x 250 mm) waste
HPLC Gem and phenomenon C18 (5μm, 270nm Sensitive, accurate, Gradient
(Xu Et Al,2014) curcumin 4.6 x 250 mm) (Gem) and precise elution, a small
420nm range of
(curcum linearity
in)
HPLC Gem C-18 (5μm, 4.6 x 250 mm) 270nm Simple, rapid stability Low
(Mastanamma Et indicating sensitivity,
Al, 2010) organic waste
HPLC Gem Enable C 18 G column 285nm Simple method, High tailing
(Kudikala Et Al, (5μm, 4.6 x 250 mm) accurate factor for Gem
2014)
HPLC Gem Kromasil (5μm, 4.6 x 150 247nm Simple method, High organic
(Devanaboyina Et mm) accurate waste
Al,2014)
HPLC Gem Intersil , C-18(5μm, 4.6 x 260nm Sensitive method High organic
(Rajesh Et Al, 150 mm) waste
2011)
HPLC Gem C 18 (3μm, 4.6 x 100mm) 276nm Sensitive method Gradient
(Lanz Et Al, 2007) elution
LC-MS-MS Gem and other --- Mass Simple and sensitive Less accurate
(Nussbaumer Et anticancer spectro method and precision
Al,2010) drugs metry
(MS-
MS)
HPTLC Gem --- 268nm Rapid stability -
(Borisagar Et Al, indicating method for
2012) HPTLC analysis
PROPOSED Gem Phenomenon luna C18 275nm Isocratic, economical, No
HPLC METHOD column (5μm, 4.6 x 250 and efficient stability quantification
mm) indicating method. of degraded
products

2.2 Aims and Objectives

This chapter aimed to develop the HPLC method with indication of stability, and which can be used to
effectively separate and quantify Gem and its metabolites by derivatization reaction. The method developed was
later applied in the optimization of Gem and its metabolite derivatives through the effects of temperature, time,
and pH of buffer. The HPLC stability indicating method would also be studied. It is important to have an
overview of Gem pharmacokinetics in cancer cells so as to have a good understanding of the mechanism of
chemo-resistance. Thus, the need for a simple and sensitive method for quantifying Gem and its metabolites. For
HPLC analysis, optimizing the composition of both the mobile phase and the stationery phase can achieve
efficient separation of compounds, and also a symmetrical peak shape. However, this strategy may not work
well with compounds that have the same chemical structure as that of Gem and its metabolites. In addition to
this, there is the challenge of weak UV absorbance that will give a less sensitive detection. In order to address
these challenges, there is need to have a better derivative technique. One important requirement for
derivatization is to have a modification of the retention times of the analytes. For instance, in RP-HPLC, to
increase the retention time of high polar compounds, we can introduce a non-polar chemical group that may
have stronger absorbance of UV [12]. The following are the specific objectives of this chapter:

i. To develop an isocratic HPLC method that can be used to quantify DNS-GEM.

ii. To use the chromatographic method to determine DNS-GEM and DNS-Cl.
iii. To investigate how DNS-GEM can be optimized under different conditions of temperature, time, and
pH buffer.
iv. To validate the HPLC method developed and obtain acceptable accuracy, precision, and linearity as
provided in ICH guidelines.
v. Develop a stable DNS-GEM in the derivatization process.

2.3 Chemicals and Reagents

We obtained Gemcitabine 99+ % from Thermo Fisher Scientific New Zealand Limited. Phosphate buffered
saline (PBS, pH 7.4), and 3, 4-ethylenedioxythiophene (EDOT) (97 %) tablets were obtained from Sigma
Aldrich, New Zealand. We purchased Fe(III)tosylate from Watson International limited, China. The solvents
and reagents used in preparing the mobile phase were of HPLC grade and analytical reagent grade respectively.
The rest of the chemicals were of reagent grade. The water that was used in the preparation of buffers was
obtained from Millipore system, through reverse osmosis of ≈ 0.22 μm Millipore.

2.4 Experimental Method

2.4.1 HPLC method for the determination of DNS-GEM and DNS-Cl

Preparation of the sample

We applied a simple and direct method of Gem derivatization using DNS-Cl because the reagent can react with
both primary and secondary amino groups to produce stable derivatives that are effectively retained on the
HPLC-UV column [9, 16]. The stock solutions for Gem and its metabolites will be prepared at a concentration
level of 200 μg/mL and stored at a temperature of 4 ºC for a later use. The preparation of the stock solutions of
the derivatization agent DNS-Cl was as follows: 2 mg of DNS-Cl was dissolved in 1 ml of acetonitrile and used
for sample derivatization. In the next step of preparation of the derivatization sample, 50μl of standard solution
of Gem and its metabolites at a concentration of 10 μg/ml, and 100μL of 2mg/mL stock DNS-Cl in acetonitrile
were transferred into a 2ml micro-centrifuge tube. The tube was then capped and put in a vortex mixing
equipment and mixed for about 1 minute. After mixing, 100μL of sodium bicarbonate/sodium carbonate buffer
(0.1 M; pH 10.5) was added into the tube. The sample was then incubated at 60 ºC for about 10 min before
centrifuging at 1400 rpm for about 5 min and transferring the supernatant into another tube. The sample was
then diluted with water in the ratio of 1:10, ready for injection into the HPLC-UV analyser system. A flow
diagram of the entire process of sample preparation is shown in figure 13 below:
 Figure 13: A flow diagram of the sample preparation process

To know the retention time of each component, three samples are prepared in the ways described as follows: (1)
matrix (water + 0.1 sodium carbonate/bicarbonate buffer + acetonitrile), (2) standard DNS-Cl, (3) reaction
derivatized Gem and metabolites. To stabilize the final volume, all the three samples were prepared at the same
volume as summarised below:

Table 5: Summary of preparation of the three samples

Samples volume Incubation at 60 OC

50μL 100μL 100μL for 10 min
1. Matrix water Acetonitrile Buffer

2. Standard DC water DC + Buffer

Acetonitrile
(2mg/ml)
3. Derivatised Gem, di Gem, di or triphosphate DC + Buffer
or triphosphate + water (10 μg/ml) Acetonitrile
(2mg/ml)

2.4.2. Optimizing the conditions of the derivatization reaction

Gem has polar molecules which have a short retention time and can be retained on the HPLC column. In
addition, Gem exhibits limited absorption in the visible or UV wavelength and lacks fluorescence at lower
concentrations. Thus, derivatization is a very necessary step for analysing Gem molecules. Each group had
varied conditions for derivatization reagent. In the current study, DNS-Cl was used as a derivatization reagent.
Studying the optimization of the reaction conditions helps to improve the retention time and sensitivity of the
HPLC. Factors such as reaction time, temperature, and pH of the medium affects the yield of DNS
derivatization. All the three conditions were investigated by preparing derivatized sample of gem at a
concentration of 10 μg/ml. The following values of pH were tested: 9, 9.5, 10, and 10.5. We varied the pH of
the samples by adding 0.4M Na2CO3. Optimizing the temperature of reaction of DNS-Cl and Gem is one
important factor in chemical reactions. The effect of temperature on the reaction of derivatized sample of Gem
was investigated within a range of 50-80 °C. Optimization of the reaction time was investigated by preparing
standard solutions of Gem that were derivatized in the time intervals of 20, 30, 40, 50, and 60 minutes, and then
HPLC analysis.

2.4.3 Development of the HPLC method for assay of DNS-GEM

A RP-HPLC method was used in quantifying DNS-GEM. The HPLC used was an Agilent series 1260 HPLC
obtained from Agilent Corporation, Germany. It comprised of a quaternary pump, an auto sampler, a vacuum
degasser, and a compartment with a thermostat and a photodiode array. Chemstation software obtained from
Agilent Corporation was used for acquiring the HPLC data. A Kinetex Evo C18 column was used (obtained
from Phenomenex, USA). The column temperature was set at 35 oC. After carrying out investigations on
different mobile phase-column combinations, one mobile phase (mobile phase A) consisted of 0.1% formic acid
in water at 4.5M, pH 4.5, and another (mobile phase B) consisted of acetonitrile + water (80+20). A linear
gradient ran 40 percent B for 4 minutes, then from 40%B to 90% B for 15 minutes. It was then held at 90%B
until 20 minutes elapsed and the system set back to its initial conditions. The total time from sample loading was
about 25 minutes with 3 minutes post-time, then the flow rate was kept at 0.4 mL/min throughout the run. At
248 nm and 264 nm, the UV detection was recorded with a bandwidth of 4 nm. A 0.45 μm filter was used to
filter the mobile phases, before degassing them by subjecting them to a sonication solution for about 10 minutes
before use. All the samples analysed had an injection volume of 20 μL.

We then validated the derivatization conditions after optimization, and the HPLC system, and then applied it in
the development of derivatization reaction for gemcitabine. Validated analytical methods to quantify DNS-GEM
are important for the success of conducting clinical and non-clinical and/or biopharmaceutics and pharmacology
studies. Validation of bioanalytical methods include performing all procedures necessary to demonstrate that a
given method used to quantitatively measure analytes is undoubtedly reliable and reproducible for its intended
use. These methods were validated for accuracy, precision, sensitivity, linearity and range, and rhobustness as
stipulated in the ICH guidelines.

2.4.4 Validation of the optimized method

2.4.4.1 Accuracy and Precision

Intra-day accuracy and precision were determined by analysing 3 replicates of 5 μg/mL, 2 μg/mL, and 3 μg/mL,
and analysing the same concentrations with 3 replicates in a period of three days. Injection repeatability
(instrumental precision) was determined by examining ten injections of a sample of gemcitabine to determine
the percentage relative standard deviation. The method accuracy was determined by comparing the
concentration obtained in the experiment, and that obtained from the true concentration.

2.4.4.2 Linearity and Range

A stock solution of gem at concentration of 200 μg /mL was prepared by dissolving in water. Serial dilutions of
the stock were then prepared using water to produce standard solutions of 5.0 to 0.5 μg/mL that were used in the
preparation of 3 replicates of the DNS-GEM sample.

2.4.4.3 Limit of detection (LOD) and limit of quantification (LOQ)

LOD and LOQ were calculated and used to determine the sensitivity of the HPLC method. The two limits were
determined by injecting lower serial concentrations of the samples to obtain a peak with signal to noise ratio of
3:1 and 10:1. To obtain LOD and LOQ, the standard deviation of the Y-intercepts of regression line were
divided by the slope, as in the following equations:

δ
LOD=3.3
slope
δ
LOQ=10
slope
Where:

δ - the standard deviation of y-intercepts of regression lines

Slope - the slope of the calibration curve
2.4.4.4 Recovery of liquid-liquid extraction

The standard samples of DNS-Cl and DNS-GEM were prepared following two methods respectively. In the first
method, the reaction was performed by adding DNS-Cl to Gem without extraction as described earlier in section
2.4.1. A similar concentration was prepared following the same process, but now methyl tert-butyl ether
(MTBE) was added into the sample in order to extract the dansyl derivatives as described in the following
paragraph:

To get rid of plastic residues, 0.5 ml aliquot of methyl tert-butyl ether (MTBE) is added into the 2.0 ml micro-
centrifuge tube. The tube will then be vortex-mixed for about 3 minutes before discarding the MTBE and the
tube left to dry. Gem standard solutions measuring 50 µL and concentration of 10 µg /ml will then be added to
the washed micro-centrifuge tube. We will then add 100 μL of 2 mg/ml of the derivatization agent (DNS-Cl) in
acetonitrile, followed by addition of 100 µL of 0.1M sodium bicarbonate/carbonate with pH of 10.5. The tube
will then be put in a vortex mixer for 3 minutes, followed by incubation at 60 oC for about 10 minutes. Once
incubation is complete, 1.0 ml of MTBE is added for extraction of dansyl-derivatives. The tube will then be
subjected to vortex-mixing and centrifugation again, before the MTBE layer is transferred to another micro-
centrifuge tube and evaporated using a stream of nitrogen at a temperature of 45 oC. Before the HPLC analysis,
the extract is reconstituted in 250 µL of water and diluted in the ratio 1:10.

2.4.5 Stability of DNS-Gem in derivatization reaction

DNS-Cl is a derivatization reagent that reacts with Gem. Gem was prepared at a concentration of 10 mg/mL,
dissolved in DNS-Cl and buffer, and then stored in a 2.0 ml micro-centrifuge tube that was covered with
aluminium foil, at a storage temperature of 4oC. The concentration of DNS-Gem in the reaction was determined
after 1 day, 5 days, 10 days, and 14 days.
2.5 Results and Discussion

2.5.1 Determination of dansylated Gem (DNS-GEM) and Dansyl Chloride (DNS-Cl) using the HPLC
method

At first, the DNS-GEM was prepared at a concentration of 10 μg/mL using the HPLC-MS since each peak can
easily be determined by mass. Mobile phase A was made of 0.1% formic acid in water (4.5 M) at pH 4.5, while
mobile phase B was made of acetonitrile + water (80+20). A linear gradient ran 10% mobile phase B for 2
minutes, then to 90% mobile phase B until after 22 minutes to eliminate substances that eluted late from the
column. The system was then initialized (see figure 14).

Figure 14: HPLC-MS showing the peaks for DNS-GEM and DNS-Cl

The same concentration of the sample was run in the HPLC-MS to determine the peaks by mass. With the
mobile phase having different linear gradients while the HPLC-UV parameters remained the same (preparation
method, same mobile phase, flow rate, and detection), the linear gradient is modified to increase DNS-OH from
8.4 min to 2.3 min. The peaks in the HPLC are libelled with their respective retention times which are indicative
of the time duration taken by a compound to come out from the column. In figure 15 (c), the DNS-GEM is
separated with no evidence of degradation peaks. At the first 2-3 minutes, the chromatogram shows some small
peaks which represent the “noise” from the injection are ignored. The larger peaks are representative of the
chemicals present in the sample. The peaks labelled 2.327 minutes, 12.921 minutes, and 13.9 minutes represent
DANS-OH, DANS-GEM, and DANS-Cl respectively. In the HPLC-UV three samples were run to confirm that
the peak labelled 13.9 minutes is the DNS-GEM. Individual DNSCL standards and matrix were injected in order
to identify these peaks (see figure 15 (a) and 15 (b)).

15(a)

[Insert your figure]

 15(b)

15(c)

Figure 15: HPLC-UV curves for….

Gem and its metabolites have polar molecules that are not easily retained on the columns of C18. In addition to
this, Gem has poor retention time on chromatography and low sensitivity in HPLC analysis at low
concentrations. There was difficulty in detecting gem at low concentrations. Gem metabolites also exhibit low
absorption in the visible or UV wavelength region, and lack florescence. The most appropriate method for
determining these metabolites is by using ion-pair chromatography. Furthermore, this method on RP-HPLC
columns provides higher efficiency compared to fixed-site ion exchanger and can also provide greater versatility
if additional secondary equilibria are exploited for optimizing the separation process [13]. Using the ion-pair for
diphosphate and triphosphate increases the polarity of phosphates as earlier discussed. It is difficult to detect
Gem using this method because it lacks a phosphate group. In IP-HPLC or RP-HPLC method, it is hard to
detect both Gem and its metabolites in one sample.

RP-HPLC

Gem

Di and Triphosphate

IP-HPLC

Gem

Di and Triphosphate

[Insert your figures]

Considering only Gem, it still exhibits poor retention and sensitivity in RP-HPLC analysis. Thus, derivatization
becomes a convenient way for analysing the drug. It is important to determine the reaction time for DNS-GEM
before optimizing the derivatization reaction.

2.5.2 Optimizing Derivatization Reaction Conditions

It is only neutral form of an amine that can react with DNS-Cl. Gem is one example of a neutral amine, and
therefore, the optimum pH should be sufficient to prevent ionization of Gem. However, the pH is controlled by
increasing side reactions, e.g. the formation of sulfonic acid at relatively high pH values. Furthermore, HCL the
derivatization reaction process releases HCl, requiring a buffer to maintain an optimum pH. DNS-Cl slightly
dissolves in water but readily soluble in acetonitrile, while Gem dissolves in water. This means that the reaction
proceeds in a mixed solvent system containing a homogenous solution of acetone/water. The reaction of DNS-
Cl and Gem was found to be dependent on the pH. If the conditions are not sufficiently alkaline, then the DNS-
derivatives will not be formed, the pH should be slightly greater than 8.5. The entire reaction can be understood
in three competing reactions: As the pH increases, the rate of DNS-GEM also increases, but this is accompanied
by increased hydrolysis of the DNS-Cl. Increasing the buffer concentration decreases the DNS-GEM, and
increases the process of DNS-Cl hydrolysis (see table 6). We also studied the buffer pH to investigate its effects
on the derivatization reaction. This was studied using 0.1M sodium carbonate/ sodium bicarbonate buffer at a
pH range of 9-10.5. We varied the pH of the solution by addition of 0.4M Na 2OH. From the results obtained, it
was shown that at pH range 9-9.5, the reaction was minimal, as compared to when the pH was raised to 10.5 and
the peak areas were stable (see figure 16). The pH 9 represented the lower area for DNS-GEM, while pH 10.5
represents the stable peak areas. Keeping the other parameters stable i.e. 60 oC for 10 minutes resulted in
insignificant difference in the yields obtained. Providing optimal conditions should lead to the most effective
reaction in a limited amount of reagent, and for this reason, pH 10.5 was taken as the optimal pH for further
analyses.

Table 6: Effect of buffer concentration

Sample Concentration Temp (°C) Ret t: 13.9 Ret t: 2.3

of buffer at
different pH DANS- DANS-OH
GEM (area)
(area)
1 0.1M pH10.5 60 355.3 2812.8
2 1M pH9.5 60 160.6 4660.7

Figure 16: Effect of pH on the reaction

We also examined different temperatures with a fixed reaction time 10 minutes to determine the optimum
temperature of the reaction for DNS-GEM. The concentrations of reagents and solvents remained constant so
that only temperature was varied. The temperature series used was 50, 60, 70, and 80oC. The yields were then
evaluated by measurement of the HPLC peak areas. It was observed that as the temperatures were raised from
50 to 80oC, so did the yields. Very low temperatures were not sufficient enough to complete the reaction, but
from 60oC, there was rapid evaporation of acetonitrile which led to bumping and solubility problem of the
reaction mixture. Thus, we used 60oC to complete the study. Finally, it was important to optimize the reaction
time between DNS-Cl and Gem. This was achieved by preparing standard solutions of DNS-Cl and Gem after
10, 15, 20, 30, 45 and 60 minutes, and then analysing the solutions in HPLC. It was observed that the area of
DNS-Gem started to decrease as the time of reaction increased. Thus, a reaction time of 10 minutes was taken as
appropriate for sufficient derivatization reaction to take place.

2.5.3 Developing the HPLC Method

There are a number of HPLC methods for the quantification of DNS-GEM that have been reported in the
literature [28, 32, 33]. Various columns were trialled, including Kinetex Evo (150 x 3 mm, 3 μm), and Gemini
(250 x 4.6 mm, 5 μm) from Phenomenex, USA. Both columns indicated almost similar retention times of about
12.7 and 13.9 minutes. Others did not show pure peaks. Locatelli et al. (2012), state that chromatographic
performance can be improved by packing columns with low diameter particles that will result in higher
efficiency, improved mass transfer, optimal velocity, and sensitivity. The disadvantage of using a stationery
phase packing with low-diameter packing is that pressure increases with the square of particle diameter. Another
important parameter for stability is the purity of the peaks which eliminate any degradation peaks in a sample.
The Kinetex column was used to achieve the optimum peak. After testing different buffer and organic solvent
combinations, a mobile phase with 30 mM acetonitrile-sodium hydrogen carbonate (NaHCO 3) at pH 10.0 was
used at a flow rate of 0.4 mL/min. The temperature of the column was optimized at 35 °C. Table 7 below shows
a summary of the optimized chromatographic parameters used in quantifying DNS-GEM.

Table 7: Optimized parameters for quantifying DNS-GEM in chromatography

Parameters Details
Column Kinetex Evo C18 (150 X 3 mm, particle size 3 μm and 100 Å)
Flow Rate 0.4 mL/min
Detection UV detector, 248 nm PDA detector, 200 to 400 nm for peak purity testing
Column 35 °C
temperature
Injection volume 20 μL
Mobile phase Mobile phase A consisted of 0.1% formic acid in water (pH 4.5, 4.5 M) and mobile
phase B was water + acetonitrile (20+80).
Linear gradient
TIME Buffer (A) ACN + water (B)
(80+20)
0 60 40
4 60 40
15 10 90
20 10 90
22 60 40

Retention time 13.9 mins

2.5.4 Validating the Optimized Method

2.5.4.1 Limit of detection (LOD) and limit of quantification (LOQ)

LOD refer to the lowest level of analyte that can easily be detected by use of analytical method, but need not to
be quantified. On the other hand, LOQ is the lowest level of analyte that can be quantified by use of analytical
method. The value of LOD was 0.3ng/mL, and that of LOQ was 0.9ng/mL [180].

2.5.4.2 Linearity and Range

In analytical methods, linearity describes the ability to find an area that is directly proportional to analyte
concentration in a peak of a sample [176].The linearity of the standard curve of Gem was in the range of 5-0.5
μg/mL (see figure 17 below). The linear equation of the line obtained from linear regression analysis is:

y=6.4036 x – 0.2284
The coefficient of relation was obtained as:
2
R =0.9999
2.5.4.3 Accuracy and Precision

In analytical methods, accuracy refers to close the test results are in relation to the actual values. In the present
work, precision refers to closeness between injections of one given sample. It can also be expressed in terms of
reproducibility, repeatability, and intermediate precision [180]. Table 8 below provides a summary of the
accuracy and precision of the results obtained. Intra-day accuracy ranged from 98.5 to 100 % with a %RSD <
4%. Inter-day accuracy ranged from 95 to 100% with %RSD < 2%. Generally, there was low variations between
the results obtained, and were all below the limits established and acceptable by regulatory authorities.

Table 8: Data showing accuracy and precision as obtained from intra- and inter-day studies of the HPLC
method

Gem concentration Intra-day Inter-day

(ng/ml) Accuracy Precision Accuracy (%) Precision (%

(%) RSD)
(% RSD)

5 99.8 3.3 100 0.6

2 100 2.6 100 1.3

0.5 98.5 2.6 95 1.7

2.5.4.4: Robustness

From the results obtained in table 8 above, it can be argued that the HPLC method developed in this study is
very robust. The %RSD of <4% is an indication of high precision despite variations in the pH of the buffer,
temperature, flow rate, mobile phase, and injection volume. Table 9 provides a summary of the effect of changes
of different chromatographic parameters on the HPLC method. The robustness data is a clear indication of how
reliable the HPLC method developed is with respect to variations in different chromatographic parameters.
Table 9: The effect of varying different parameters on the RP-HPLC method of DNS-GEM

Parameters Conditions Accuracy (%) Precision (% RSD)

Mobile Phase Buffer 60 % ACN 40 % 100.3 1.2

Buffer 61 % ACN 39 % 98.5 0.6

Buffer 62 % ACN 38 % 97.7 0.6

Flow Rate 0.3 mL/min 97.7 0.6

0.4 mL/min 101.1 1.9

0.5 mL/min 99.3 3.7

Column 30oC 99.8 3.4

Temperature
35oC 102.7 0.9

40oC 104.5 3.9

Injection Volume 10 µL 99.8 0.9

20 µL 101.9 2.8

30 µL 105.3 3.3

2.4.4.5 Recovery

Methods that have been published for analysing Gem include: liquid-liquid extraction [15, 16, and 19], direct
injection extraction [5], solid phase extraction with UV or mass spectrometric detection [6], and protein
precipitation [3, 4, 7, 16, and 17]. Liquid-liquid extraction is a versatile technique with various advantages
depending on the analyte. The technique is helpful in minimizing baseline noise and increase sensitivity. From
previous observations, it is evident that chloroform could be replaced by MTBE in popular solvent systems
applied in liquid-liquid extraction. This technique was inspired by the classical methods of Folch and Bligh &
Dyer (1959) who utilized the principles of liquid-liquid extraction because of varying densities of liquids which
made the upper and lower phases of the solvent system. Samples in our study were extracted in a 1-phase
system which ensured optimal contact between the biological material and the extraction solvent. A significant
difference between the chloroform/methanol-based Folch and Bligh & Dyer procedures and the MTBE lipid
extraction procedure is the opposite order of the organic phase and the aqueous phase (see figure 17). The upper
phase which contains lipid is formed by MTBE and methanol because both solvents have low densities
(Methanol: 0.79 g/mL, MTBE: 0.74 g/mL) compared to water. As a result, the organic phase is collected prior to
coming into contact with non-extractable residues or the aqueous phase at the bottom of the tube.

Figure 16: Chloroform/methanol-based Folch and Bligh & Dyer procedure and the MTBE lipid extraction
procedure.
Compared to chloroform, MTBE is more polar solvent and has a water solubilizing capacity as high as 1.4%
without requiring phase separation. Subsequently, the lipid organic phase becomes higher in water content,
which in turn improves the extraction efficiency for acidic lipids.

Recovery of lipid was achieved in the MTBE phase. First, the extraction cells treated with Gem (the biological
material) is solubilized with DNS-Cl and buffer before incubation at a temperature of 60 oC for the reaction to
proceed. The MTB is then added to the crude reaction sample so that phase separation can be induced. Free
Gem – a polar molecule that does not react with DNS-Cl moves into the water phase, while lipid molecules
remain in the organic MTBE phase (see figure 17).

Figure 17: Separation of lipids and free Gem in MTBE solvent

Three concentrations of Gem standards for preparation of DNS-Gem were extracted, and using HPLC, their
recoveries were determined based on internal sample of known gem standard concentrations (see table 10). The
recoveries achieved by adding MTBE, and without adding MTBE were different depending on the concentration
of Gem, which varied from 0.5 to 5.0 μg/mL. At a Gem concentration of 0.5 μg/mL, it was difficult to determine
the area after 1 mL of MTBE solvent was added. In accordance with Nernst’s distribution law, molecules of
lipid distribute between the two immiscible liquids in such a way that the ratios of the two liquid phases remain
constant despite the addition of MTBE. In this case, increasing the amount of MTBE increases the % recovery.
However, it can be seen from table 10 that the addition of 1.0 mL or 2.0 mL of MTBE does not bring a
significant change in % recovery.

Table 10: Percent recovery of lipids before and after sample extraction by addition of 1 mL MTBE and 2.0 mL
MTBE.

Gem Area Area Recovery %

concentration (Before (After extraction)
(μg/ml) extraction)
1.0 mL of 2.0 mL of 1.0 mL of 2.0 mL of
MTBE MTBE MTBE MTBE
5 15.2 10.8 11.1 71.0 73.0
2 4.2 2.8 3.0 66.6 71.4
0.5 1.0 --- 0.6 --- 60.0

2.5.5 Stability of Gem in derivatization reaction

The stability of Gem in the product samples of DNS-GEM was evaluated by re-injection of the samples on the
HPLC column from an immediate run following storage at room temperature, and at 4 oC prior to assessment.
The HPLC conditions were as follows: Mobile phase A was made of 0.1% 4.5M formic acid in water at pH 4.5,
and mobile phase B was made of acetonitrile + water (80+20) with a linear gradient, flow rate was 0.4 mL/min,
C18-luna column, and a UV detector at 246 and 264 nm. While at room temperature, the samples were only
stable for 3 days. The samples still had accuracy and precision within acceptable limits (< 15%) and there was
no change in the sensitivity of DNS-GEM. At 4 oC, the samples were stable for up to 6 days, with an average of
99.6 ± 2.5 % Gem after the 6 days. The sample peak was still pure even after the 6-day storage at 4 oC.