Original Article
Anal Chem Ind J, Volume: 17( 1)

Validated Isocratic/Gradient RP-HPLC for Simultaneous Estimation of Paracetamol Ibuprofen and Caffeine in Marketed Formulations Using Diclofenac as Internal Standard

*Corresponding author:
Dushyanth Reddy V Department of Pharmaceutical Chemistry, KLE's University College of Pharmacy, KLE University, Belagavi 590001, India
Tel: 08312471399; E-mail: [email protected]

Received: October 16, 2016 Accepted: January 16, 2017 Published: January 22, 2017

Citation: Palled PJ, Dushyanth Reddy V, Mannor VS, et al. Validated Isocratic/Gradient RP-HPLC for Simultaneous Estimation of Paracetamol Ibuprofen and Caffeine in Marketed Formulations Using Diclofenac as Internal Standard. Anal Chem Ind J. 2017;17(1):116.

Abstract

Objective: To develop a validated RP-HPLC method for simultaneous estimation of Paracetamol Ibuprofen and Caffeine. Methods: The HPLC instrument used was Shimadzu LC-20AD with reverse phase ODS-Hypersil-C18 (250 mm × 4.6 mm, 5 μm) column using Acetonitrile:water (90:10) pH adjusted to 2.8 as mobile phase. The flow rate was maintained at 0.3 ml/min and UV detection was carried at 203 nm.

Results: The method was validated for linearity, accuracy, precision, specificity, robustness and ruggedness according to ICH guidelines. The retention time for Paracetamol, Ibuprofen and Caffeine was found to be 9.7, 12.66 and 10.48 respectively. The regression analysis showed good linearity over the concentration range of 1.25 μg/ml to 20 μg/ml for paracetamol, 0.625 μg/ml to 10 μg/ml for Ibuprofen and 0.625 μg/ml to 10 μg/ml for Caffeine. The recovery studies of the method gave good results in the range of 99.89% to 100.48% with less than 2% of RSD.

Conclusion: The method was found to be suitable for the analysis of marketed formulation in presence of other excipients.

Keywords

Paracetamol; HPLC; Chromatography; Ibuprofen; Anti-inflammatory

Introduction

Paracetamol (Acetaminophen) is chemically N-(4-hydroxyphenyl) acetamide is a crystalline solid is a sparingly soluble compound which is classified under antipyretic analgesics. Drugs Classified under this class possess analgesic and antipyretic activity but lacks anti-inflammatory effects indicated for use in patients who are sensitive to aspirin with usual adult dosage 325 mcg to 650 mcg dose greater than 2.6 g/day are not advisable for prolonged treatment owing to its hepatotoxicity. Antipyretic effect of acetaminophen affords to the inhibition of endogenous leukocytic pyrogens released from cells upon external stimuli or upon activation with exogenous pyrogen. acetaminophen possesses analgesic activity in arthritis and musculoskeletal disorders.

Acetaminophen is available in various formulations say suppositories, tablets, capsules, granules and solutions. Ibuprofen is chemically 2-(4-isobutylphenyl) propionic acid is a crystalline solid is a sparingly soluble compound. It is classified as Non-Steroidal anti-inflammatory drug it was the first NSAID approved after Indomethacin. First NSAID to become over the counter (OTC) drugs. It is marketed as racemic mixture even its biological response owed almost evidently with S-(+) isomer. Ibuprofen is more potent than aspirin but less effective than indomethacin. Ibuprofen produces moderate levels of gastric irritation [1]. ibuprofen is indicated in patients suffering with rheumatoid arthritis, osteoarthritis, fever and dysmenorrhea. Caffeine is chemically 1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione acetamide is a crystalline solid is a sparingly soluble compound which is chemically methyl xanthines naturally occur in coffee (coffee Arabica) which is generally termed as stimulant and as a bronchodilator. caffeine is generally added to other OTC analgesic and stimulants.

Methodology

1. UV Profiling of API's

2. HPLC Method Development

a. Isocratic

b. Gradient

3. Selection of Internal Standard

4. Assay Methodology for Quantification of API's

Chromatography

Ultraviolet spectroscopy (absorption spectroscopy) is one of the most widely used technique for quantification of organic and inorganic molecules. Chromatography is a technique for separation of organic molecules. Hyphenated instrumentation technique say (HPLC-UV/LC-MS/LC-NMR) is a technique to reconcile output abnormalities of one instrument with input abnormalities of other instruments. Lower edge or conventional instruments say UV/Fluorescence/IR serve as detectors in high end or Hyphenated instrumentation technique say (HPLC-UV/LC-MS/LC-NMR). High Performance Liquid Chromatography is a technique of separation of molecules (in μg-ng/mL) in shorter time. Various literature reported HPLC methods are available for simultaneous estimation of paracetamol and ibuprofen either alone or in combination with other API's but very few direct HPLC method for simultaneous estimation of paracetamol, ibuprofen and caffeine according our knowledge. We employ a criteria of standard addition method for inclusion of internal standard in quantification of API's.

UV profiling of API's

As the current research work is executed with HPLC with UV detector (Dual Wavelength detector), we had performed spectral scan for all the API's of interest both individually and simultaneously. Our aim was to identify an isobestic point (wavelength at which all API's have common absorbance). Identification of Isobestic point using UV spectroscopy is the criteria for wavelength selection in HPLC-UV. In UV scan of API's Paracetamol, Ibuprofen and Caffeine (absorption maxima as a function of wavelength) Figures. 1 and 2 isobestic point was found to be 203 nm. The same wavelength was selected in HPLC-UV(LC-20-AD).

analytical-chemistry-Absorbance-maxima

Figure 1: Absorbance maxima as a function of wavelength.

analytical-chemistry-Absorbance-maxima-Caffeine

Figure 2: Absorption maxima of Caffeine and isobestic point.

HPLC Method Development

Mobile phase selection

Various combinations of mobile phases with and without buffer at varied pH from 2-8, with more emphasis on final pH of mobile phase is adjusted to ± 2 units of pka value of API. Various combinations of mobile phase tried are highlighted in Table.

Column or stationary phase selection

Various stationary phases (columns) have been screened for current research, the best fit column with respect to tailing factors and theoretical plates, eventually ODS Hypersil C18 was selected for analysis.

Materials, reagents and chemicals

Paracetamol, Ibuprofen and Caffeine pure drug was obtained as gift sample from Sun Pharma Sparc Research Centre Vadodara. The other chemicals like methanol, acetonitrile and milli-Q waters were procured from Milli pore water system available at BSRC. The tablet dosage forms imol plus procured from local market [2].

Instrumentation

UV instrument consist of Shimadzu UV, PH meter, Milli pore water purification system. The HPLC system consist of Shimadzu LC 20AD, Hamilton injector of 20 μl capacity and detected by SPD-20A UV detector with LC-Solutions software. The stationary phase used is reverse phase ODS-Hypersil-C18.

Chromatographic conditions

The mobile phase consists of Acetonitrile:water (90:10) pH adjusted to 2.8, all the solvents used in analysis are filtered through 0.45 μ Sartorius filter paper, degassed by ultra-sonication for 10 min. The flow rate was maintained at 0.3 ml/min. Aliquots of the samples (20 μl) (injection volume) were injected and the total run was kept at 20 min. the chromatogram was monitored at 203 nm.

Standard solutions and Calibration curves

The standard stock solutions of the drug was prepared by dissolving 10 mg pure drug in 100 ml of mobile phase. Serial dilutions were made from stock solutions using acetonitrile.

Sample preparation

Ten tablets of Paracetamol, Ibuprofen and Caffeine were weighed and grinded into fine powder in mortar and pestle. A mass of powder equivalent to 100 mg of Paracetamol, Ibuprofen and Caffeine was accurately weighed and transferred to a volumetric flask containing acetonitrile. The resultant solution was sonicated for 5 min and filtered through nylon filter and the volume was made up to 100 ml using acetonitrile. serial dilutions were made from the stock solution of formulation, and were analyzed for their recovery studies [3].

Method validation

The method was validated according to the ICH guidelines for the following parameters.

Linearity and sensitivity

Standard solutions of Paracetamol, Ibuprofen and Caffeine was prepared in the concentration range of 5 μg/ml to 25 μg/ml. Then 20 μl of each solution were injected in triplicate on to the column and the chromatogram was developed using above mobile phase Acetonitrile:water (90:10) pH adjusted to 2.8 ratio. The RF values were plotted against the corresponding concentration to obtain the calibration graph. The LOD and LOQ were calculated based on the equation: LOD=3.3 × A/B and LOQ=10 × A/B. Where, A is SD of peak areas of the drugs taken as a measure of noise and B is the slope of corresponding calibration curve Figures. 3- 5.

analytical-chemistry-Absorbance-Chromatogram

Figure 3: Chromatogram of paracetamol linearity graph of paracetamol.

analytical-chemistry-Ibuprofen-Chromatogram

Figure 4: Chromatogram of Ibuprofen linearity graph of Ibuprofen.

analytical-chemistry-Ibuprofen-Caffeine

Figure 5: Chromatogram of Caffeine linearity graph of Caffeine.

Precision

The interday and intraday precision studies were conducted by using three different concentrations of the standard (initial, medium and final concentrations) in triplicate in a day and on three consecutive days.

Accuracy

The accuracy of the method was examined by performing recovery studies in triplicate using standard addition method (50%, 100% and 150%). Accurately known amount of sample was added to a known amount of pre-analyzed tablet powder and was analyzed (Table 1).

Parameter Paracetamol Ibuprofen Caffeine
Retention time (Rt) 9.7 12.66 10.48
Linearity range (µg/ml) 1.25 µg/ml to 20 µg/ml 0.625 µg/ml to 10 µg/ml 0.625 µg/ml to 10 µg/ml
Regression equation y=61963x + 11807 y=31198x +77914 y=59842x +42403
Limit of detection (µg/ml) 0.28481225 0.187937934 0.264438458
Limit of quantification (µg/ml) 0.94937417 0.626459779 0.881461526
Regression coefficient (r2) 0.999 0.999 0.999

Table 1. Linearity parameters for calibration curve.

Paracetamol Caffeine Ibuprofen
Peak Area Conc. RSD Peak Area Conc. RSD Peak Area Conc. RSD
888147 1.25 0.69258 783034 1.25 0.442707 470798 1.25 0.692588
1659642 2.5 0.53228 1527003 2.5 0.69224431 869036.667 2.5 0.53228782
3197167 5 0.36195 3035632 5 0.709403 1645744 5 0.361959

Table 1a. Accuracy.

S.no Intra Day Precession Inter day precession
Paracetamol Ibuprofen Caffeine Paracetamol Ibuprofen Caffeine
1 6385180 3189497 5990397 6385180 3189497 5990397
2 6394967 3179829 6011626 6394967 3179829 6011626
3 6498615 3218876 6057253 6498615 3218876 6057253
4 6401950 3184222 6014400 6400632 3186556 5960127
5 6342337 3204056 5944931 6366615 3256463 5917302
6 6400594 3223011 5978122 6394706 3199781 5987988
AVG 6403941 3199915.167 5999455 6406786 3205167 5987449
SD 51392.4 18266.60881 38005.54 46554.44 28573.27 47187.29
RSD 0.802512 0.570846659 0.633483 0.726643 0.891475 0.788103

Table 1b. Precision.

Robustness

Robustness of the method was determined by introducing small changes in the mobile phase composition, change in flow rate and detection wavelength. During initial stages of development of method, the method was subjected to small changes and the effect of small changes in method on the detection of Paracetamol, Ibuprofen and Caffeine with respect to peak shape, RT values and stability were studied (Tables 2-5).

Mobile
Phase
Flow Theoretical plates Tailing factor
AVG STDEV RSD AVG STDEV RSD
90 : 10 0.5 12323.978 56.20974539 0.456100663 0.93933333 0.00305505 0.325236032
90 : 10 0.7 9067.419667 78.84922682 0.869588369 0.888 0.00953939 1.074255857
88 : 12  0.5 15478.77167 135.7386882 0.876934495 0.99166666 0.00709459 0.715421736
88 : 12 0.7 11609.59933 154.6095267 1.331738695 1.021 0.00754983 0.739454891
92 : 08 0.5 5950.82 40.23684315 0.67615628 0 0 0
92 : 08 0.7 4445.731 49.73684297 1.118755115 0 0 0

Table 2. Robustness study for Paracetamol.

Mobile
Phase
 Flow Theoretical plates Tailing factor
AVG STDEV     AVG STDEV
 90 : 10  0.5 12864.37067 116.9338918 90 : 10 0.5 12864.37067 116.9338918
 90 : 10 0.7 11470.76967 130.9244533 90 : 10 0.7 11470.76967 130.9244533
 88 : 12  0.5 14257.88433 209.0422122 88 : 12 0.5 14257.88433 209.0422122
 88 : 12 0.7 12586.42633 104.8210764 88 : 12 0.7 12586.42633 104.8210764
 92 : 08  0.5 12718.54433 71.83204801 92 : 08 0.5 12718.54433 71.83204801
 92 : 08 0.7 11747.39667 135.0314692 92 : 08 0.7 11747.39667 135.0314692

Table 3. Robustness parameters of Ibuprofen.

Mobile
Phase
Flow Theoretical plates Tailing factor
AVG STDEV RSD AVG STDEV RSD
 90 : 10  0.5 15841.41867 104.1076266 0.657186258 1.31 0.007549834 0.576323239
 90 : 10 0.7 12461.14467 201.2005414 1.614623269 1.361 0.02433105 1.787733293
 88 : 12  0.5 16618.58767 209.3578273 1.259781105 1.2573 0.011590226 0.921810109
 88 : 12 0.7 13637.39533 201.6091045 1.478354918 1.324 0.012529964 0.94637191
 92 : 08  0.5 14483.07133 84.91817942 0.58632715 0 0 0
 92 : 08 0.7 12089.129 67.05188783 0.554646144 0 0 0

Table 4. Robustness parameters of Caffeine.

 Ruggedness study for Paracetamol Ruggedness study for Ibuprofen Ruggedness study for Caffeine
 S.NO Analysit-1 Analyst-2 Analyst-1 Analyst-2 Analyst-1 Analyst-2
1 Peak area 3208599 3240878 1641232 1686064 3051201 3020092
2 Peak area 3193454 3148553 1652496 1699056 3044639 3116484
3 Peak area 3189449 3238949 1643503 1681409 3011056 3097548
  AVG 3197167 3209460 1645744 1688843 3035632 3078041
  STDEV 10100.61 52755.83 5956.917 9145.834 21534.85 51070.9
  RSD 0.315924 1.64376 0.361959 0.541544 0.709403 1.659201

Table 5. Ruggedness.

Results and Discussion

Optimization of the RP-HPLC method

Various solvent systems were evaluated to obtain better chromatogram. Initially, methanol, HPLC grade water, acetate buffer, acetonitrile and phosphate buffer were tried in different ratios [4]. But the resolution was not satisfactory. Finally, the mobile phase consisting of Acetonitrile:water (90:10) pH adjusted to 2.8 found to be optimum.

Linearity and sensitivity

The linearity was evaluated by determining six working standard solutions containing 5 μg/ml to 25 μg/ml of Paracetamol, Ibuprofen and Caffeine in triplicate with good correlation coefficient in the concentration range. (r=0.989). The LOD and LOQ was found to be 0.0532 μg/ml and 2.344 μg/ml respectively Figures. 6- 8.

analytical-chemistry-Baseline-Caffeine

Figure 6: Chromatogram without Baseline Correction with Baseline Correction.

analytical-chemistry-internal-standard

Figure 7: Chromatogram without internal standard Chromatogram with internal standard.

analytical-chemistry-Assay-standard

Figure 8: Chromatogram of Standard Assay Chromatogram of Assay of Formulation.

Assay

The amount of Paracetamol, Ibuprofen and Caffeine present per tablet was calculated by comparing the peak area, with that of the internal standard.

Selection and role of internal standard

Conventional quantification techniques carry an error, to minimize the error in analysis, recovery and quantification of API was done with respect to internal standard method. criteria for selection of internal standard; structurally similar compounds have similar retention times, diclofenac is used as internal standard [5]. The conventional quantification technique (using linearity and regression is obsolete and carry certain amount of random error. Internal standard analysis always carry standard amount of determinate error throughout the analysis.

The structures of API's were drawn using Chem. Draw Ultra-10

Assay Methodology

Recovery experiment

To study the accuracy, reproducibility and the precision of the proposed method recovery experiments were carried out. A fixed amount of pre-analyzed sample was taken and standard drug was added at three different concentrations and each level was repeated for 3 times. The recovery was estimated to be more than 100% [6].

Validated gradient RP-HPLC method for estimation of Paracetamol, Caffeine and Ibuprofen

Stationary phase: C18 250 mm × 4.6 mm, 5 μ, Inertsil ODS 3V.

Mobile phase A: 6.8 gm of KH2PO4 in 1L Milli-Q water pH adjusted to 3 with Orthophosphoric acid

Mobile phase B: Filtered and degassed Acetonitrile (HPLC grade).

Detector: UV at wavelength 230 nm.

Flow rate: 1 mL/min (Run Time 30mins)

Injection volume: 20 μl

Mode: Gradient

Elution order: Paracetamol, Caffeine and Ibuprofen.

Retention time: Paracetamol,Caffeine and Ibuprofen is 6, 8 and 17 min (Table 6), Figures. 9 and 10.

analytical-chemistry-Assay-paracetamol

Figure 9: HPLC (Gradient) Chromatogram of paracetamol, Ibuprofen and Caffeine.

analytical-chemistry-Ibuprofen-paracetamol

Figure 10: HPLC (Isocratic) chromatogram of Paracetamol, Ibuprofen and Caffeine.

Time KH2PO4 Buffer pH 3.0 Acetonitrile
0.01 85 15
5 85 15
12 25 75
20 25 75
22 85 15
30 85 15

Table 6. Gradient programming.

Preparation of samples and standard stock solution A: Weigh accurately 130 mg of Paracetamol, 16 mg Caffeine, and 80 mg Ibuprofen into a 50 ml volumetric flask. Add sufficient amount of methanol, sonicate to dissolve, cool and dilute up to the mark with methanol. The above stock solution was further diluted 5 ml to 100 ml with methanol [6].

Standard stock solution B: Weigh accurately 136.5 mg of Paracetamol, 16.8 mg Caffeine, and 84 mg Ibuprofen into a 50 ml volumetric flask. Add sufficient amount of methanol, sonicate to dissolve, cool and dilute up to the mark with methanol. The above stock solution was further diluted 5 ml to 100 ml with methanol.

Sample stock solution: Twenty tablets were weighed accurately and finely powdered. The powder equivalent to 1 tablet was taken in a 50 ml volumetric flask, methanol was added and sonicate for 20 minutes, cool and further diluted up to the mark with methanol. The above stock solution was further diluted 2 ml to 100 ml with methanol. Then filtered through 0.45 μ membrane filter.

Placebo for Paracetamol: Weigh accurately 0.135 gm of placebo taken into 50 ml volumetric flask. Add 16 mg Caffeine, 80 mg Ibuprofen and sufficient amount of methanol, sonicate for 20 minutes, cool and dilute up to the mark with methanol. The above solution further diluted 5 ml to 100 ml with methanol. Then filtered through 0.45 μ membrane filter.

Placebo for Caffeine: Weigh accurately 0.135 gm of placebo taken into 50 ml volumetric flask. Add 130 mg Paracetamol, 80 mg Ibuprofen and sufficient amount of methanol, sonicate for 20 minutes, cool and dilute up to the mark with methanol. The above solution further diluted 5 ml to 100 ml with methanol. Then filtered through 0.45 μ membrane filter.

Placebo for Ibuprofen: Weigh accurately 0.135 g of placebo taken into 50 ml volumetric flask. Add 130 mg Paracetamol, 16 mg Caffeine and sufficient amount of methanol, sonicate for 20 min, cool and dilute up to the mark with methanol. The above solution further diluted 5 ml to 100 ml with methanol. Then filtered through 0.45 μ membrane filter (Tables 7-10).

Sample Name Peak area
Paracetamol
Retention time Area
Caffeine
Retention time Peak area of Ibuprofen Retention time Similarity factor
Paracetamol/Ibuprofen/Caffeine
STD A 7557233 6.32 564849 8.54 2016114 17.58  
STD A 7580906 6.31 566807 8.53 2020175 17.57  
STD A 7588646 6.31 567043 8.52 2022898 17.57  
STD A 7587502 6.31 567267 8.52 2021357 17.57  
STD A 7584143 6.30 566700 8.49 2019249 17.57  
STD A 75944237 6.30 567367 8.49 2021417 17.56  
STD B 7568881 6.31 565933 8.50 2016610 17.56 0.98
RSD 0.17% 0.11% 0.16% 0.22% 0.12% 0.03%  

Table 7. Data of specificity test for Paracetamol.

Parameter Acceptance Paracetamol Caffeine Ibuprofen
Theoretical Plates NLT 2000 7771 10581 135129
Tailing Factor NMT 2 1.0 1.1 1.1
Capacity Factor NLT 2 1.5 2.4 6.0
Similarity Factor  0.98 to 1.02 0.98 0.98 0.98
%RSD of STD A for Area NMT 2 0.17% 0.16% 0.12%
%RSD of STD A for RT NMT 2 0.11% 0.22% 0.03%
%RSD of STD for Area NMT 2 0.17% 0.18% 0.20%
%RSD of STD for Retention time NMT 2 0.11% 0.22% 0.04%

Table 8. System Suitability.

Sample Name Paracetamol Caffeine Ibuprofen
 Area Amount
mg/tab
% Label Claim  Area Amount
mg/tab
% Label Claim  Area Amount
mg/tab
% Label Claim
1 7402044 318.8 98.1 550272 39.4 98.6 2019414 200.1 100.1
2 7424325 319.8 98.4 552223 39.6 99.0 2026164 200.8 100.4
3 7437356 320.5 98.6 553173 39.7 99.2 2028301 201.1 100.6
4 7485967 322.8 99.3 554184 39.8 99.4 2033877 201.8 100.9
5 7550710 325.6 100.2 558013 40.1 100.1 2042587 202.7 101.4
6 7552770 325.2 100.1 557624 40 99.9 2042212 202.3 101.2
SD   2.881 0.886   0.229 0.573   0.972 0.486
% RSD   0.89 0.89   0.58 0.58   0.48 0.48

Table 9. Repeatability of Paracetamol Ibuprofen and Caffeine.

            Parameter Acceptance Paracetamol Caffeine Ibuprofen
%RSD of Assay NMT 2 0.89 0.58 0.48
Similarity Factor 0.98 to 1.02 0.98 0.98 0.98

Table 10. Summary of repeatability.

Linearity

Standard solution (50% Level): Weigh accurately 32.50 mg of Paracetamol, 4.02 mg Caffeine, and 21.0 mg Ibuprofen into a 50 ml volumetric flask. Add sufficient amount of methanol, sonicate to dissolve, cool and dilute up to the mark with methanol. The above stock solution was further diluted 2 ml to 100 ml with methanol.

Standard solution (60% Level): Weigh accurately 39.3 mg of Paracetamol, 4.83 mg Caffeine, and 25.5 mg Ibuprofen into a 50 ml volumetric flask. Add sufficient amount of methanol, sonicate to dissolve, cool and dilute up to the mark with methanol. The above stock solution was further diluted 2 ml to 100 ml with methanol.

Standard solution (80% Level): Weigh accurately 52.80 mg of Paracetamol, 46.42 mg Caffeine, and 34.1 mg Ibuprofen into a 50 ml volumetric flask. Add sufficient amount of methanol, sonicate to dissolve, cool and dilute up to the mark with methanol. The above stock solution was further diluted 2 ml to 100 ml with methanol.

Standard solution (100% Level): Weigh accurately 64.90 mg of Paracetamol, 8.06 mg Caffeine, and 40.5 mg Ibuprofen into a 50 ml volumetric flask. Add sufficient amount of methanol, sonicate to dissolve, cool and dilute up to the mark with methanol. The above stock solution was further diluted 2 ml to 100 ml with methanol.

Standard solution (120% Level): Weigh accurately 77.30 mg of Paracetamol, 9.62 mg Caffeine, and 48.5 mg Ibuprofen into a 50 ml volumetric flask. Add sufficient amount of methanol, sonicate to dissolve, cool and dilute up to the mark with methanol. The above stock solution was further diluted 2 ml to 100 ml with methanol.

Standard solution (140% Level): Weigh accurately 90.0 mg of Paracetamol, 11.18 mg Caffeine, and 56.5 mg Ibuprofen into a 50 ml volumetric flask. Add sufficient amount of methanol, sonicate to dissolve, cool and dilute up to the mark with methanol. The above stock solution was further diluted 2 ml to 100 ml with methanol.

Standard solution (150% Level): Weigh accurately 32.50 mg of Paracetamol, 4.02 mg Caffeine, and 21.0 mg Ibuprofen into a 50 ml volumetric flask. Add sufficient amount of methanol, sonicate to dissolve, cool and dilute up to the mark with methanol [7]. The above stock solution was further diluted 2 ml to 100 ml with methanol (Tables 11-20).

  Sample Name    Level       Paracetamol Caffeine Ibuprofen
Peak Area Conc. Response Factor Peak Area Conc. Response Factor Peak Area Conc. Response Factor
1 50% 64188 64.80 59372.5 4731 8.01 35378.8 17414 41.92 24958.2
2 60% 77398 78.36 59118.8 5712 9.63 35481.5 20893 50.90 24582.5
3 80% 103953 105.28 59075.1 7684 12.80 35888.8 27871 68.07 24504.7
4 100% 127640 129.41 59158.1 9468 16.07 35319.7 34086 80.84 25257.3
5 120% 152373 154.13 59279.0 11336 19.19 35405.6 40589 96.81 25130.7
6 140% 177636 179.46 59290.9 13263 22.30 35612.5 47167 112.78 25049.0
7 150% 189707 192.02 59194.8 14203 24.07 35291.7 50228 119.77 25123.2
SD       105.75     208.79     288.66
% RSD       0.18%     0.59%     1.16%

Table 11. Linearity of API's at various spiking levels.

Name %Y Intercept Correlation coefficient Response Factor (%RSD)
Paracetamol -0.1 1.000 1.18%
Caffeine 0.4 1.000 0.59%
Ibuprofen -1.6 1.000 1.16%
Acceptance -2 to +2 NLT 0.99 NMT 5%

Table 12. Summary of linearity.

Sample Name Paracetamol Caffeine Ibuprofen
Peak Area Amount mg/tab %Label Claim Peak Area Amount mg/tab %Label Claim Peak Area Amount mg/tab %Label Claim
 1 7406357 326.6 100.5 552989 39.9 99.8 2073280 199.8 99.9
2 7338309 323.4 99.5 552303 39.9 99.7 2077918 200.2 100.1
 3 7360016 324.4 99.8 553631 40 99.9 2084750 200.8 100.4
SD   1.61 0.49   0.04 0.12   0.51 0.25

Table 13. Robustness (Low pH).

            Parameter Acceptance Paracetamol Caffeine Ibuprofen
% Label Claim 95% to 105% 99.9 99.8 100.1
Similarity Factor 0.98 to 1.02 0.99 0.98 1.01

Table 14. Summary of Robustness (low pH).

Sample Name Paracetamol Caffeine Ibuprofen
Peak Area Amount mg/tab %Label Claim Peak Area Amount mg/tab %Label Claim Peak Area Amount mg/tab %Label Claim
 1 7364772 321.1 98.8 554575 40 100.0 2046088 200.8 100.4
2 7400597 322.8 99.3 555880 40.1 100.3 2055765 201.9 100.9
 3 7369187 321.6 99.0 554293 40 100.1 2045168 201 100.5
SD   0.86 0.26   0.05 0.14   0.56 0.28
RSD   0.27 0.27   0.15 0.15   0.28 0.28

Table 15. Robustness (High pH).

Parameter Acceptance Paracetamol Caffeine Ibuprofen
% Label Claim 95% to 105% 99.0 100.1 100.6
Similarity Factor 0.98 to 1.02 1.00 0.99 1.00

Table 16. Summary of robustness (high pH).

Parameter Acceptance Paracetamol Caffeine Ibuprofen
% Label Claim 95% to 105% 99.4 99.0 99.0
Similarity Factor 0.98 to 1.02 0.98 0.98 1.00

Table 17. Summary of robustness (low flow rate).

Sample Name Paracetamol Caffeine Ibuprofen
Peak Area Amount mg/tab %Label Claim Peak Area Amount mg/tab %Label Claim Peak Area Amount mg/tab %Label Claim  
 1 8197386 323.5 99.5 617630 39.7 99.3 2282308 198.6 99.3  
2 8172205 322.2 99.1 615123 39.5 98.8 2273102 197.6 98.8  
 3 8184580 323 99.4 615999 39.6 99.0 2275068 197.9 99.0  
SD   0.66 0.20   0.10 0.25   0.50 0.25  
RSD   0.20 0.20   0.25 0.25   0.26 0.26  

Table 18. Robustness (Low flow rate).

Sample Name Paracetamol Caffeine Ibuprofen
Peak Area Amount mg/tab %Label Claim Peak Area Amount mg/tab %Label Claim Peak Area Amount mg/tab %Label Claim  
 1 6668504 322.6 99.3 498142 39.6 99.0 1837498 198.6 99.3  
2 6673044 322.9 99.4 498715 39.7 99.2 1838632 198.8 99.4  
 3 6639102 321.3 98.9 496605 39.5 98.7 1831277 198 99.0  
SD   0.84 0.26   0.08 0.20   0.40 0.20  
RSD   0.26 0.26   0.21 0.21   0.20 0.20  

Table 19. Robustness (High flow rate).

Parameter Acceptance Paracetamol Caffeine Ibuprofen
% Label Claim 95% to 105% 99.2 99.0 99.2
Similarity Factor 0.98 to 1.02 0.98 0.98 0.98

Table 20. Summary of robustness (High flow rate).

Accuracy

Requirements

Standard solution (Recovery 50% level): Weigh accurately 162.5 mg of Paracetamol, 20 mg Caffeine, and 100 mg Ibuprofen into a 50 ml volumetric flask. Add sufficient amount of methanol, sonicate to dissolve, cool and dilute up to the mark with methanol. The above stock solution was further diluted 2 ml to 100 ml with methanol.

Standard solution (Recovery 100% level): Weigh accurately 325 mg of Paracetamol, 40 mg Caffeine, and 200 mg Ibuprofen into a 50 ml volumetric flask. Add sufficient amount of methanol, sonicate to dissolve, cool and dilute up to the mark with methanol. The above stock solution was further diluted 2 ml to 100 ml with methanol [8].

Standard solution (Recovery 150% level): Weigh accurately 487.5 mg of Paracetamol, 60 mg Caffeine, and 300 mg Ibuprofen into a 50 ml volumetric flask. Add sufficient amount of methanol, sonicate to dissolve, cool and dilute up to the mark with methanol. The above stock solution was further diluted 2 ml to 100 ml with methanol (Tables 21-25).

  Level Paracetamol Caffeine Ibuprofen
Peak Area Amount mg/tab %Label Claim Peak Area Amount mg/tab %Label Claim Peak Area Amount mg/tab %Label Claim
50% 3816748 101 0.22 288181 103 0.73 1050819 101 0.12
100% 7573648 99 0.14 562381 100 0.31 2035228 101 0.08
150% 11261934 99 0.03 839915 100 0.15 2991330 99 0.17

Table 21. Recovery table of Paracetamol, Caffeine and Ibuprofen.

Parameter Acceptance Criteria Paracetamol Caffeine Ibuprofen
Recovery 50% 98% to 102% 101 103 101
Recovery 100% 98% to 102% 99 100 101
Recovery 150% 98% to 102% 99 100 99
%RSD of Recovery NMT 2 0.03 0.15 0.17
Similarity Factor 0.98 to 1.02 1.00 0.99 1.00

Table 22. Summary of accuracy.

  Sample Name Paracetamol Caffeine Ibuprofen
Peak Area Conc. Response Factor Peak Area Conc. Response Factor Peak Area Conc. Response Factor
1 6960251 320.8 98.7 544569 39.7 99.2 544569 39.7 99.2
2 7113910 323.7 99.6 550444 39.6 99.0 550444 39.6 99.0
3 6944705 319.3 98.2 543543 39.5 98.8 543543 39.5 98.8
4 7138815 324.8 99.9 552295 39.7 99.3 552295 39.7 99.3
5 7117815 323.8 99.6 550579 39.6 99.0 550579 39.6 99.0
6 6931597 319 98.2 543636 39.6 98.9 543636 39.6 98.9
SD   2.51 0.77   0.07 0.19   0.07 0.19
% RSD   0.78 0.78   0.20 0.20   0.20 0.20

Table 23. Intermediate Precision Data for Paracetamol, Ibuprofen and caffeine.

            Parameter Acceptance Paracetamol Caffeine Ibuprofen
%RSD of Assay NMT 2 0.78 0.20 0.18
% Variation NMT2 0.1 0.3 1.5
Similarity Factor 0.98 to 1.02 0.98 0.99 1.00

Table 24. Summary of intermediate precision (Ruggedness).

  Paracetamol   Caffeine   Ibuprofen
  Area mg/tab %
Label
Claim
%
relative
differ
Area mg/tab %
Label Claim
 %
relative
differ
Area Amount mg/tab %Label Claim %
relative
differ
 
0 7411567 322.6 99.3 0.0 553845 39.4 98.6 0.0 2052065 201.2 100.6 0.0  
2 7436506 323.7 99.6 0.3 555885 39.6 99.0 0.4 2069954 202.9 101.5 0.9  
4 7434897 323.6 99.6 0.3 555679 39.6 98.9 0.3 2067476 202.7 101.3 0.8  
8 7445787 324.1 99.7 0.5 556076 39.6 99.0 0.4 2067943 202.7 101.4 0.8  
12 7446004 324.1 99.7 0.5 556104 39.6 99.0 0.4 2063880 202.3 101.2 0.6  
16 7419306 322.9 99.4 0.1 554280 39.5 98.7 0.1 2058591 201.8 100.9 0.3  
20 7386753 321.5 98.9 0.3 552865 39.4 98.4 0.2 2055739 201.5 100.8 0.2  
24 7461670 324.7 99.9 0.7 556740 39.7 99.1 0.5 2063585 202.3 101.1 0.6  

Table 25. Solution stability of Paracetamol Ibuprofen and Caffeine.

Orthogonal analysis

API's from other sources need to be characterized by conventional as well as orthogonal testing's as described in ICH Q6B [9]. Additional testing should include parameters or method to determine the suitability of the reference material not necessarily captured by the drug substance (e.g., more extensively in tracing related substance impurities).

Conclusion

The switch over of two different methods say Isocratic/gradient analysis for the same sample ensured greater resolution of analyte of interest in comparison with isocratic in gradient mode, and there is no relative substance impurities detected by variation in the method confirming the purity of the samples and the method is validated according to ICH 21 CFR guidelines and the method is robust in both isocratic and gradient mode.

References