RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR

SIMULTANEOUS ESTIMATION OF PARACETAMOL AND

ALPRAZOLAM IN BULK AND PHARMACEUTICAL DOSAGE FORMS

Presented by
Mrs. S. DURGA MOUNIKA
Reg. No. 167N1S0401
(2016– 2018)

Under the Guidance of

Dr. M. NARENDER., M. Pharm., Ph.D.,
Associate Professor

VIJAYA INSTITUTE OF PHARMACEUTICAL SCIENCES FOR WOMEN

Enikepadu, Vijayawada

1
August, 2018
DEPARTMENT OF PHARMACEUTICAL ANALYSIS & QUALITY ASSURANCE
 Contents
1. INTRODUCTION

2. REVIEW OF LITERATURE

3. DRUG PROFILE

4. MATERIALS AND METHODS

5. RESULTS AND DISCUSSION

6. CONCLUSION

7. REFERENCES

2
 Introduction

• Analytical research and development is a requisite part of the pharmaceutical industry.

• In this present work a new RP-HPLC method was developed for the simultaneous estimation of

Paracetamol (PAR) and Alprazolam (ALP) in bulk and pharmaceutical dosage forms.

• PAR and ALP were commercially available as combination dosage forms used in the treatment of

anxiety, depression and cold.

• Commonly available combined tablet dosage forms includes STS from Emcure Labortaories which

contains (500 mg of PAR and 0.25 mg of ALP).

• PAR as pure standard reference drug was obtained from Dr. Reddy’s Laboratory, Hyderabad and

ALP as pure standard reference drug was obtained from Cipla Laboratory.
3
 AIM & OBJECTIVE:

In the present study, an attempt was made to provide a simple and accurate RP-HPLC method for

the simultaneous estimation of PAR and ALP in bulk and pharmaceutical formulations, and further

to validate the established method according to ICH Q2 (R1) guidelines.

PLAN OF WORK:

The experimental work has been planned in a stepwise manner to develop simple, rapid and selective

analytical method for estimation of PAR and ALP using RP-HPLC as follows:

Step 1:

 Study of literature and procurement of materials.

 Study of physico-chemical properties of drug such as solubility determination of PAR and ALP in

4 various solvents.
  Determination of absorption maxima of drugs in UV-Visible region.

 Preparation of standard and sample solutions of PAR and ALP.

 Development of simple RP-HPLC method for the simultaneous estimation of PAR and ALP in

combined dosage form.

 Selection of stationary phase and mobile phase.

 Optimization of chromatographic conditions.

Step 2:

Validation of the developed method in accordance with the analytical validation parameters

such as linearity, accuracy, precision, robustness, system suitability, limit of detection

(LOD), limit of quantification (LOQ) etc., as per ICH (Q2R1) guidelines.

5
 Review of Literature

 PAR and ALP were commercially available as combination dosage forms in the treatment of anxiety,

depression and cold. Commonly available combined tablet dosage forms (PAR+ALP) includes STS

(Emcure Labortaories).

 Literature survey reveals that development of various Spectrophotometric [Kumar et al., 2011; Rele et

al., 2016] and High Performance Liquid Chromatography (HPLC) methods [Chauhan et al., 2015;

Parva et al., 2015; Rele et al., 2016] for estimation of PAR and ALP in various dosage forms in

individual and in combination with other drugs. However, there was a UV-Spectrophotometric method

[Unnisa et al., (2014)] for the simultaneous estimation of PAR and ALP in tablet dosage forms. To the

best of our literature survey there is no method developed for the HPLC analysis of PAR and ALP in

their combined dosage forms.

6
 Analytical methods could be wet-chemical and instrumental type. Instrumental methods are crucial in

new analytical method development with help of advanced and more sensitive instruments. HPLC is

one of specific and sensitive chromatographic technique routinely used for drug analysis.

High performance liquid chromatography (HPLC):

• HPLC consists of a liquid mobile phase which is pumped under pressure through a

stainless steel column.

• Seperation of particles is based on the relative affinities of molecules towards the stationary

phase.

• Solute molecules that are more affinity towards stationary phase elute later and and lesser affinity gets

eluted faster.

• Once the effluents were separated the monitoring can be carried out with a variety of detectors.
7
 Advantages of HPLC:
 It is extremely quick and efficient.

 It is accurate and highly reproducible.

 Repetitive reproducible analysis can be done by using the same column.

 It provides a high degree of selectivity for specific analyses

 It delivers higher resolution.

 It is versatile and extremely precise when it comes to identifying and quantifying chemical

components.

 It is largely automated and hence basic HPLC runs can be performed with minimal training.

8
HPLC METHOD DEVELOPMENT
 Flow chart of crucial steps involved in HPLC method development:

9
 Parameters for method validation:

• Specificity is the ability to assess clearly the analyte in the presence of components
Specificity which may be expected/targeted to be present.

• The linearity of an analytical procedure is its ability (within a given range) to obtain
Linearity the test results which are directly proportional to the concentration (amount) of analyte
in the sample

• The accuracy of an analytical procedure expresses the closeness of agreement between

Accuracy
the values

• The precision of an analytical procedure is usually expressed as the variance,

Precision standard deviation or coefficient of variation of a series of measurements

• The robustness is the measurement of its purity capacity to remain unaffected by

Robustnes
s small, but deliberate variations in the method parameters

• LOD and LOQ are the lowest amount of drug present in tha analytical sample that is
LOD & detected and quantified
LOQ
10
 Drug Profile

Paracetamol Alprazolam
Generic name: Paracetamol Alprazolam

Label claim: 500 mg. 0.25 mg

Category Analgesics and antipyretics. Anxiolytics

Common name: N-(4-hydroxyphenyl) acetamide. 8-Chloro-methyl-6-phenyl-4H [1,2,4]
triazo [4,3a][1,4] benzodiazepine.

Chemical structure:

Molecular formula C8H9NO2. C17H13C1N4

Molecular weight 151.163 g/mol. 308.769 g/mol.

Properties It’s a white crystalline solid which is It is white to off white solid crystals.
odourless and bitter in taste.

Melting point 336-342 0F. 228-229.5 0C.

11
Half life 24 hrs 11.2 hrs
 Freely soluble in water, alochol ethanol etc. Soluble in methanol. Ethanol
Solubility Insoluble in water
Insoluble in Benzene
Pharmacokinetics Rapidly absorbed by oral
Absorption Rapidly absorbed by the GI tract. administration

Distribution Its volume of distribution is roughly 50 L 80% ALP binds to human serum
protein
Metabolism PAR is metabolised primarily in the liver Extensively metabolized by
cytochrome P450 3A4
Excretion Excreted by kidneys Excreted by kidneys

Mechanism of action It is generally a weak inhibitor of synthesis of It binds non specifically to

prostaglandins (PGs). benzodiazepine receptors BNZ1 and
BNZ2.

Dosage forms Tablets, syrups and suppositories Tablets

Dosing For adults and children > 12 years = 500- For adults = 0.25 -0.5 mg thrice a
1000 mg every 4-6 hrs day.
Side effects Allergic reactions, Flushing, Low blood Ataxia, head ache, sedation, weight
pressure, fast heart beat etc. loss, memory impairment etc.
Contraindications To those who are hypersensitivity to PAR Hypersensitivity patients
12
Storage In a air tight containers at room temperature Room temperature.
 Methodology

 In the RP-HPLC method development and validation of PAR and ALP all the protocols were performed

by using different types reagents and materials. The solvents utilized were HPLC grade and all the

chemicals were analytical grade.

 Cyber lab HPLC system accomplished with UV-detector, Quantitative HPLC was performed on an

isocratic mode using Cap Cell Pack C18 column with 20 μL injection of sample loop. The output signal

was monitored and integrated using Cyber lab LC 100 software.

Preparation of Standard Stock Solutions:

Preparation of Paracetamol and Alprazolam Standard Solution:

Transfer 500 mg of PAR and 25 mg of ALP into 70 mL of diluent and sonicate the resultant solution for

15 mins and make the volume 100ml.From the above solution take 10ml and make upto 100ml with
13
diluent which is injected into the Chromatographic column.
 RP-HPLC method development (Trials):
Aliquots of the mixed solutions containing PAR and ALP were prepared and a number of trials (trails
1- 4) were performed.

Trail - 1 Trail - 3
Mobile phase: methonol: water (80:20 %v/v ) Mobile phase: ACN : Phosphate buffer (80:20 %v/v )

Trail -2 Trail - 4
Mobile phase: ACN: Phosphate buffer (60:40 %v/v ) Mobile phase: ACN: water (80:20 %v/v )

14
 Optimized method:

Preparation of mobile phase:

A combination of ACN and water (80:20 % V/V) was prepared, mixed and then degassed in ultra sonic

cleaner for 15 mins.

Diuent/Blank:

Use mobile phase as diluent and blank.

Preparation of standard mixture of PAR and ALP: Transfer 500 mg of PAR and 25 mg of ALP

into 70 mL of diluent. Resulted solution sonicated for 15 mins and the volume made up to 100 mL with

diluent. From the above solution pipette out 10 ml into 100 ml volumetric flask and make up the final

volume with the diluents to get final concentrations (500 µg/mL) and (25µg/mL) , respectively.

15
 Method validation

Validation is a process which provides high degree of assurance that activity will consistently

produce a desired result to meet its predetermined specifications and quality characteristics.

System suitability: Transfer 500 mg of PAR, and 25 mg of PAR into 70 mL of diluent. Resulted

solution sonicated for 15 mins and the volume made up to 100 mL with diluents. From the above solution

pipette out 10 ml into 100 ml volumetric flask and make up the final volume with

Validation parameters includes:

Specificity: Solutions of standard and sample were prepared as per the test method and injected

into chromatographic system.

Linearity: A series of solutions of standard drug substance were prepared in the concentration

ranging from 50-175 µg/mL for PAR and 0.25-1.5 µg/mL for ALP.
16
Accuracy: The accuracy was carried out by adding known amounts of standard drug to the analyte (three

concentrations levels 50, 100 and 150 % of the labeled claim.

Precision: Precision was determined in terms of repeatability. System precision and method precision was

established in accordance with ICH guidelines.

 The system precision was determined by analyzing the standard solution of PAR and ALP.

 The method precision was determined by analyzing the samples of PAR and ALP.

Robustness: Robustness of the method was evaluated by assaying test solutions under slight but deliberate

changes in analytical conditions, such as change in flow rate, change in wave length and change in pH of

buffer.

 Flow rate change: In this experiment the flow rate has changed as 0.8 ml/min, 1 ml/min and 1.2 ml/min.

 Wavelength change: In this experiment the test samples were analyzed at the wavelength of 225 nm, 230 nm

and 235 nm.

17
 LOD: A working standard solution of PAR and ALP was prepared to the lower concentrations of

linearity range from the standard stock solutions and injected into the chromatographic column.

The following formula was applied for LOD,

LOD = 3.3 × D/S

LOQ :The parameter LOQ was determined on the basis of height of the signal and noise (10:1)

of the response at concentration below the linearity range. The following formula was applied

for LOQ,

LOQ = 10 × D/S

18
 Results and discussion
 Selection of UV detection wavelength: The standard solutions of PAR and ALP prepared were
scanned over the wavelength region of 200 – 400 nm (UV region). Based on the peak
absorption maxima of analytes 236 nm was selected as the absorption wavelength.

• Preparation of Standard stock solutions of PAR and

ALP: Transfer 10 mg of PAR and 10 mg of ALP into 10

mL volumetric flask and add 7 mL of diluent. Resulted
solution sonicated for 15 mins and the volume made up to
10 mL with diluent to get final concentrations (1000
µg/mL). 1 mL of above solutions transfered into 10 mL
volumetric flasks and made the volume up to the mark to
get the final concentration of 100 µg/mL. Finally it was
scanned under UV spectrophotometer to know the
absorbance maxima.
19
 Optimized method for RP-HPLC for PAR and ALP:

The optimized conditions were applied for development of analytical method for PAR and ALP. The Cap

cell pack C18 column (250 x 4.6 mm, 5μ) was used because of its advantage of high resolving capacity,

better reproducibility, low-back pressure and low tailing.

Optimized Chromatographic conditions

Parameters Chromatographic conditions

Column Cap cell pack C18 column ( 250 x 4.6 mm, 5 μ)
Mobile phase (Ratio) ACN: Water (80:20 % V/V)
Elution mode Isocratic
Flow rate 1 mL/min
Detection wavelength 236 nm
Injection volume 20 µL
Run time 10 min
Temperature 25°C
20
 Observation:

The elution of analytes was achieved for maintaining run time 10 mins, the peaks were eluted at

4.8 and 6.2 min for PAR and ALP, respectively.Good retention time with sharp peak was

observed. No tailing or fronting was seen for both the analytes.

Rt of PAR= 4.8
Rt of ALP= 6.2

Optimized Chromatogram

21
  Assay of PAR and ALP:

HPLC Chromatogram of Standard mixture. HPLC Chromatogram of Sample

Assay results for optimized method

Tablet formulation Label claim per % Drug found % RSD

tablet (mg) ± SD (n=6)
STS: PAR (500 mg) PAR-500 mg 100.9 1.1
+ ALP (0.25 mg) ALP- 0.25 mg 101.1 1.7
22
 System Suitability:
The column was equilibrated with the mobile phase for 30 min prior to the injection of the drug
solution. A working standard solution prepared was injected five times into the HPLC system

Injection - 1

Injection - 2

23
 Injection - 3

Injection - 4 Injection - 5
24
 Data of System Suitability for PAR & ALP

Retention time Peak area Tailing factor Theoretical plate

S. No (mins) (mV) (N)
PAR ALP PAR ALP PAR ALP PAR ALP
1 4.80 6.17 159899.2 16282.4 1.58 1.39 7219 6788
2 4.80 6.17 158416.4 16492.1 1.63 1.22 7528 6524
3 4.80 6.17 156936.2 16326.3 1.52 1.24 7458 6899
4 4.80 6.17 155446.6 16442.8 1.49 1.48 7654 6652
5 4.80 6.17 156374.5 16455.6 1.56 1.32 7412 7519
Mean 4.80 6.17 157414.5 16455.4 - - - -
S.D - - 1757.87 178.25 - - - -
%RSD - - 1.11 1.08 - - - -

Observation:
From the system suitability studies it is observed that all the parameters are within
25
limit
 Specificity:. The chromatograms of standard and sample were identical with nearly same

retention time hence it was concluded that the standard and sample are same and there was no

interferences .

Chromatogram of Standard Chromatogram of Sample

Chromatogram of Blank

26
  Linearity:

Chromatogram of PAR (50 µg/mL) &ALP (0.25 µg/mL).

27
Chromatogram of PAR (75 µg/mL) & ALP (0.5 µg/mL).
 Chromatogram of PAR (100 µg/mL) & ALP (0.75 µg/mL).

28 Chromatogram of PAR (125 µg/mL) & ALP (1.0 µg/mL).

 Chromatogram of PAR (150 µg/mL) & ALP (1.25 µg/mL).

29 Chromatogram of PAR (175 µg/mL) & ALP (1.5 µg/mL).

 Linearity Data of PAR and ALP

Concentration Peak area

Analyte drug Linear regression Equation
(µg/mL) (mV)
50 108091.1
PAR 75 158168.2 y=2001.x+3970
100 202992.6 r² = 0.999
125 253359.8
150 300806.6
175 355607.7
0.25 10828.9
ALP 0.5 14441.4 y =2549.2x+305.
0.75 16143.5 r² = 0.999
1 23161.7
1.25 30102.7
1.5 32293.1
A calibration curve was plotted against amount of drug (µg/mL) v/s chromatographic peak area
30 (mV).
 Calibration Curve for PAR (50- 175 µg/mL).

31
Calibration Curve for ALP (0.25 – 1.5 µg/mL).
  Accuracy:

Chromatogram of sample with 50% Standard addition

32
Chromatogram of sample with 100% Standard addition
 Chromatogram of sample with 150% Standard addition.

Observation:

The accuracy was expressed in terms of percent analyte recovered by the proposed method.

From the accuracy studies, the recovery value of pure drug was found in the range of 98.0 - 102.0%

which indicates that the method was accurate.

33
 Accuracy data of PAR

S. No. Spiked Sample area Sample % Recovery % Mean

level height

50% 78345.3 20101 100.5

1 50% 79368.8 20612 100.5 100.4

50% 76543.4 20794 100.4

100% 157348.2 26670 100.6

2 100% 156832.4 26871 100.9 100.7

100% 156348.4 26538 100.8

150% 225036.2 32538 99.07

3 150% 226036.2 32901 100.6 99.9

34 150% 220146.5 32799 100.2

 Accuracy data of ALP

S.No Spiked Sample area Sample % Recovery % Mean

level height

50% 295162 35021 100.8

1 50% 293853 35683 99.8 100.1

50% 293345 35942 99.8

100% 576369 70042 100.9

2 100% 579209 70861 101.1 100.5

100% 570405 70932 99.5

150% 864543 105882 99.1

3 150% 868347 105936 99.8 99.5

150% 869543 105856 99.7

35
  Precision
System precision and method precision was established in accordance with ICH guidelines.

System precision:

Injection - 1

Injection - 2

36
 Injection - 3 Injection - 4

37
Injection - 5 Injection - 6
 System Precision data of PAR and ALP

Retention time Peak area Peak height

Injection (mins) (mV)
PAR ALP PAR ALP PAR ALP
1 4.80 6.20 159899.2 15365.3 19725 1124
2 4.80 6.20 158581.7 15812.2 19736 1156
3 4.80 6.20 157476.1 15925.4 19563 1148
4 4.80 6.20 155500.2 15244.8 19856 1165
5 4.80 6.20 158891.6 15669.2 19256 1183
6 4.80 6.20 154948.8 15753.4 19985 1179
Mean 4.80 6.20 157549.6 15628.38 19686 1159
S.D - - 1765.01 266.70 - -
%RSD - - 1.1 1.7 - -

38
  Method precision:

Injection - 1

Injection - 2

39
 Injection - 3 Injection - 4

40
Injection - 5 Injection - 6
 Method Precision data of PAR and ALP

PAR ALP
Injection
RT Peak area RT Peak area
Injection-1 4.80 168581.7 6.20 17509.1

Injection-2 4.80 163512.6 6.20 17581.8

Injection-3 4.80 162918.2 6.20 17567.5

Injection-4 4.80 163441.3 6.20 17881.2

Injection-5 4.80 164682.5 6.20 17639.1

Injection-6 4.80 165910.1 6.20 17911.7

Mean 4.80 164841.06 6.20 17695.06

SD - 2125.17 - 172.12

% RSD - 1.2 - 0.97

41
  Robustness

Rt(PAR)=5.10
Variation in Flow rate
Rt(ALP)=7.22

Chromatogram of Robustness variation in flow rate (0.8 mL/min) Low.

Rt(PAR)=4.8
Variation in Flow rate
Rt(ALP)=6.2

42
Chromatogram of Robustness variation in flow rate (1 mL/min) Original.
 Rt(PAR)=4.19
Rt(ALP)=4.99
Variation in Flow rate

Chromatogram of Robustness variation in flow rate (1.2 mL/min) High.

Variation in wavelength
Rt(PAR)=4.8
Rt(ALP)=6.2

43 Chromatogram of Robustness variation in wavelength (234 nm) Low.

Variation in wavelength

Chromatogram of Robustness variation in wavelength (236 nm) Original.

Variation in wavelength

44 Chromatogram of Robustness variation in wavelength (238 nm) High.

 Robustness data of developed method - PAR

Changes in the

Parameter Chromatographic Area 1 Area 2 Mean Std Dev %RSD

conditions

0.8 (Low) 181482.2 182491.4 181986.8 713.61 0.39

Flow rate 1 (Original) 153032.4 154216.2 153624.3 837.35 0.54

(mL/min) 1.2 (High) 130605.8 131425.4 131015.6 580.11 0.44

234 nm (Low) 178091.4 177812.3 177951.8 197.31 0.11

Wavelength (nm) 236 (Original) 155862.2 156918.2 156390.3 746.49 0.47

238 (High) 165935.8 166219.1 166077.4 200.32 0.12

45
 Robustness data of developed method - ALP

Changes in the

Parameter Chromatographic Area 1 Area 2 Mean Std Dev %RSD

conditions

0.8 (Low) 15653.5 15389.2 15521.3 106.88 1.20

Flow rate 1 (Original) 17819.3 17624.5 17721.9 137.85 0.77

(mL/min) 1.2 (High) 16623.2 16824.6 16723.9 142.41 0.85

234 (Low) 16682.9 16931.4 16807.1 175.53 1.04

Wavelength (nm) 236 (Original) 17648.4 17919.5 17783.9 192.01 1.07

238 (High) 14629.6 14821.2 14725.4 135.48 0.92

46
 • LOD

Chromatogram of LOD.

LOD data of PAR and ALP

S.No Drug Peak area LOD (µg/mL)

1. PAR 31979.8 11.2

2. ALP 10567 0.24

47
  LOQ

Chromatogram of Limit of Quantification.

LOQ data of PAR and ALP

S. No Drug Peak area LOQ (µg/mL)

1. PAR 147847.7 50

2. ALP 13677.2 0.25

48
 Results of method validation and assay of PAR and ALP.

Parameters Results Acceptance criteria

PAR ALP

Specificity - - No Interference

Linearity 0.999 0.999 r2 =0.999

Accuracy 100.7 100.5 98 -102 %

Precision 1.2 0.97 RSD < 2%

Robustness <2% <2% RSD < 2%

LOD 11.2 0.24 Signal to noise ratio should

be more than 3:1

LOQ 50 0.25 Signal to noise ratio should

be more than 10:1

49 Assay 100.7% 100.5% 98 -102%

 Summary
 RP-HPLC separation was achieved on Cell Pack C18 column (250 mm × 4.6 mm, 5 µ particle

size).

 Method involves the combination of ACN and water as mobile phase in the ratio of 80:20

%V/V.

 The elute detection was monitored at 236 nm using UV-detector.

 The flow rate was at 1.0 mL/min with the sample injection volume of 20 µL.

 The separation for PAR and ALP were achieved at 4.8 and 6.2 mins, respectively.

 The system suitability parameters such as theoretical plate number and tailing factor were

found to be 7242, 1.56 for PAR and 6755, 1.15 for ALP.

 Linearity was observed in the concentration range of 50-175 µg/mL and 0.25-1.5 µg/mL for

PAR and ALP, respectively.

50
  The method was found to be accurate and the % recovery was found to be 99-101% and

99-102% for PAR and ALP, respectively.

 The correlation coefficient (r2) for PAR and ALP was found to be 0.999 for both drugs.

 LOD was found to be 11.2 µg/mL for PAR and 0.24 µg/mL for ALP.

 LOQ was found to be 50 µg/mL for PAR and 0.25 µg/mL for ALP.

 The proposed method was validated by employing these parameters according to ICH

guidelines and can be used for routine analysis.

51
 Conclusion
A simple, accurate, rapid, sensitive and precise RP-HPLC method has been developed for the

simultaneous estimation of PAR and ALP in tablet dosage form using UV-detector. A RP Cell Pack

C18 column (250 mm × 4.6 mm, 5 µ particle size) with mobile phase consisting of ACN and water

in the ratio of 80:20 % V/V was used for separation and at 236 nm.

The developed method was found to be satisfactory with good precision, linearity and accuracy. The

optimized method was validated according to ICH (Q2 R1) guidelines and all the results lie within

the specified limits.

Hence the proposed new RP-HPLC method was found valid, can be applied for routine analysis of

simultaneous estimation of ALP and PAR in their combined pharmaceutical formulations.

52
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