Quality by design (QbD) refers to the achievement of certain predictable quality with desired and predetermined specifications. The objective of this study was to develop and demonstrate an integrated multivariate approach to develop and quantify the constituent concentrations of glipizide (GPZ) drug in its pure and tablet forms. The method was developed using Zorbax Extend C-18 (50 mm × 4.6 mm × 1.8
Quality by design (QbD) is a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management [
Glipizide (GPZ), chemically known as N-[2-[4-[[[(Cyclohexylamino) carbonyl] amino] sulfonyl]phenyl]ethyl]-5-methylpyrazine carboxamide] (Figure
Structure of glipizide.
GPZ is included in the United States Pharmacopoeia [
According to literature survey, there are quite a few publications on UPLC method development [
The primary objective of this study was to implement QbD approach to develop and validate an RP-UPLC method that could separate drug in the bulk, formulated forms and human urine with in-depth understanding of the method and build in the quality during the method development to ensure optimum method performance over the lifetime of the product with a suitable degradation data.
Pure active ingredient sample of GPZ was kindly supplied by Bal Pharma, Bangalore, India, as gift. GPZ-containing tablets, Dibizide (5 mg) (Micro Labs Limited, Hosur, India) and Glynase-5 (5 mg) (USV Limited, Aurangabad), were procured from the local market. HPLC grade acetonitrile was purchased from Merck Specialities, Mumbai, India. Potassium dihydrogen orthophosphate, benzene, and orthophosphoric acid were from Qualigens, India. Doubly distilled water was used throughout the investigation.
Waters (Waters Chromatography Division, Milford, MA, USA) AQUITY UPLC system with a tunable UV detector was used for the determination of GPZ. Empower 2 software was used to record and evaluate the data collected during and following chromatographic analysis. Shimadzu Pharmaspec 1700 UV/Visible spectrophotometer was used for the initial absorbance measurement. The chromatographic separation was achieved on a Zorbax Extend C-18 (
In order to establish whether the analytical method and the assay were stability indicating, pure active pharmaceutical ingredient (API) of GPZ was subjected to stress under various conditions to conduct forced degradation studies [
Stock solution of GPZ (1 mg/mL) was prepared in diluent and it was stored at 5°C until use.
Working solutions containing 0.005–300
Fifty numbers, each of Glynase and Dibizide tablets (each tablet contained 5.0 mg GPZ), were weighed and powdered. Tablet powder equivalent to 20 mg of GPZ was transferred into 100 mL volumetric flasks and 60 mL of the mobile phase were added. The solution was sonicated for 20 min to achieve complete dissolution of GPZ, made up to the mark with mobile phase and then filtered through 0.22
A 1.00 mL of 0.05 M hydrochloric acid was added to 0.50 mL of urine, resulting in a pH of 3. The mixture was extracted with 3.00 mL of benzene in a 12 mL glass tube, which was shaken gently for 15 min. After centrifugation for 5 min, the organic phase was transferred to a conical tube for evaporation to dryness under a stream of a well-ventilated fume chambers. The residue was redissolved in mobile phase and an aliquot of 2
The method development consisted of two phases: the first is screening and the second is optimisation.
Solubility of the drug is the main criteria for any method to develop and then the UV response of the product at varying wavelengths. GPZ was found soluble in a mixture of acetonitrile and phosphate buffer. A 200
Screening: summary of method selection*.
S. no. | Column | Observations | Remarks |
---|---|---|---|
1 | Acquity BEH C8 (100 × 2.1) mm, 1.7 µm | Asymmetrical peak with fronting and tailing | Not satisfactory |
2 | Acquity BEH C18 (100 × 2.1) mm, 1.7 µm | Asymmetrical peak with tailing | Not satisfactory |
3 | Acquity HSS cyano (50 × 2.1) mm, 1.7 µm | Split peak | Not satisfactory |
4 | Acquity HSS BEH shield RP18 (50 × 2.1) mm, 1.7 µm | Asymmetrical peak | Not satisfactory |
5 | Zorbax Extend C-18 (50 × 4.6) mm, 1.8 µm | Symmetrical peak | satisfactory |
*By keeping one column constant, the mobile phase, temperature, buffer, and sample concentration parameters were changed.
UV absorption spectra of glipizide.
Zorbax Extend C-18 (
Final optimization of pH with Zorbax Extend C-18 (
S. no. | Trails taken | Observations | Remarks |
---|---|---|---|
1 | ACN : buffer (pH 3.5) (40 : 60% v/v) | Peaks found symmetrical | Satisfactory |
2 | ACN : buffer (pH 4) (40 : 60% v/v) | Peak eluted before 1 min with less theoretical plates | Not satisfactory |
3 | ACN : buffer (pH 3) (40 : 60% v/v) | Broad peak | Not satisfactory |
4 | ACN : buffer (pH 5) (40 : 60% v/v) | Broad peak and late elution | Not satisfactory |
Method optimization at different pH conditions.
ACN: buffer (pH 3.5) (40 : 60% v/v)
ACN: buffer (pH 4.0) (20 : 80% v/v)
ACN: buffer (pH 2.2) (20 : 80% v/v)
ACN: buffer (pH 5.5) (20 : 80% v/v)
Column: Zorbax Extend C-18 ( Oven temp.: 25°C. Mobile phase: ACN: buffer (pH 3.5) (40 : 60% v/v). Run time: 5 min. Flow rate: 0.2 mL/min. Diluent: mobile phase. Injection volume: 2 Blank: diluent. Wavelength: 220 nm.
The described method for the assay of GPZ was validated as per the current ICH Q2 (R1) guidelines.
Linearity and regression parameters with precision data.
Parameter | Value |
---|---|
Linear range, µg mL−1 | 0.005–300 |
Limits of quantification (LOQ), µg mL−1 | 0.005 |
Limits of detection (LOD), µg mL−1 | 0.001 |
Regression equation | |
Slope ( |
11701.20 |
Intercept ( |
5660.74 |
Correlation coefficient ( |
0.99999 |
Results of accuracy study (
Concentration of GPZ injected, µg mL−1 | Intraday | Interday | ||
---|---|---|---|---|
Concentration of GPZ found, µg mL−1 | REa, % | Concentration of GPZ found, µg mL−1 | RE, % | |
100 | 101.34 | 1.34 | 99.47 | 0.53 |
200 | 203.12 | 1.56 | 202.21 | 1.11 |
300 | 302.12 | 0.71 | 303.45 | 1.15 |
aRelative error.
Results of precision study.
Concentration injected (µg mL−1) | Mean area ± SD | RSDa | Mean Rt ± SD | RSDb |
---|---|---|---|---|
Intraday precision ( |
||||
100 | 1165237 ± 2333 | 0.20 | 2.13 ± 0.006 | 0.26 |
200 | 2347961 ± 16728 | 0.71 | 2.13 ± 0.006 | 0.28 |
300 | 3482365 ± 23172 | 0.67 | 2.13 ± 0.002 | 0.10 |
| ||||
Concentration injected (µg mL−1) | Mean area ± SD | RSDa | Mean RT ± SD | RSDb |
| ||||
Interday precision ( |
||||
100 | 1161904 ± 3535 | 0.30 | 2.14 ± 0.018 | 0.82 |
200 | 2347961 ± 16728 | 0.71 | 2.13 ± 0.006 | 0.29 |
300 | 3488820 ± 7850 | 0.23 | 2.14 ± 0.020 | 0.91 |
aRelative standard deviation based on peak area.
bRelative standard deviation based on retention time.
Results of method robustness and ruggedness study.
Condition | Modification | Mean peak area ± SD | % RSD | Mean Rt ± SD | % RSD | Theoretical plates ± SD | % RSD | Tailing factor ± SD | % RSD |
---|---|---|---|---|---|---|---|---|---|
Actual | — | 2351828 ± 11571 | 0.49 | 2.130 ± 0.005 | 0.24 | 2968 ± 5.16 | 0.17 | 1.01 ± 0.005 | 0.47 |
Temperature | 24°C | 2346161 ± 14060 | 0.60 | 2.131 ± 0.004 | 0.19 | 2962 ± 20.00 | 0.68 | 1.10 ± 0.008 | 0.76 |
26°C | 2345161 ± 16987 | 0.72 | 2.131 ± 0.004 | 0.20 | 2963 ± 16.02 | 0.54 | 1.10 ± 0.01 | 0.50 | |
Mobile phase composition (acetonitrile : buffer) | 35 : 65 | 2350328 ± 13836 | 0.59 | 2.129 ± 0.005 | 0.24 | 2960 ± 24.01 | 0.81 | 1.11 ± 0.005 | 0.47 |
45 : 55 | 2351828 ± 11572 | 0.49 | 2.131 ± 0.004 | 0.19 | 2970 ± 4.08 | 0.14 | 1.10 ± 0.004 | 0.370 | |
Flow rate | 0.22 mL/min | 2346828 ± 14139 | 0.60 | 2.133 ± 0.005 | 0.25 | 2965 ± 12.11 | 0.41 | 1.14 ± 0.008 | 0.72 |
0.18 mL/min | 2343495 ± 15290 | 0.65 | 2.132 ± 0.006 | 0.26 | 2968 ± 4.90 | 0.17 | 1.11 ± 0.006 | 0.57 | |
Wavelength | 219 nm | 2348495 ± 13198 | 0.56 | 2.132 ± 0.005 | 0.24 | 2970 ± 4.00 | 0.13 | 1.10 ± 0.006 | 0.57 |
221 nm | 2351802 ± 11535 | 0.49 | 2.129 ± 0.005 | 0.23 | 2960 ± 24.42 | 0.82 | 1.10 ± 0.004 | 0.37 | |
Analyst 1, column 1, |
2350541 ± 11350 | 0.48 | 2.129 ± 0.005 | 0.23 | 2961 ± 14.95 | 0.50 | 1.11 ± 0.008 | 0.76 | |
Analyst, column, day | Analyst 2, column 2, |
2349495 ± 10475 | 0.45 | 2.129 ± 0.005 | 0.24 | 2964 ± 15.23 | 0.51 | 1.10 ± 0.005 | 0.47 |
Analyst 3, column 3, |
2353178 ± 12027 | 0.51 | 2.135 ± 0.010 | 0.47 | 2968 ± 6.98 | 0.24 | 1.11 ± 0.010 | 0.99 |
Chromatograms obtained for placebo blank and tablet extract.
Placebo blank
Tablet extract
A 200
Results of determination of GPZ in formulations and statistical comparison with the reference method.
Formulation brand namea | Nominal amount, mg | % GPZ foundc ± SD |
|
|
|
---|---|---|---|---|---|
Reference method | Proposed method | ||||
Dibizidea | 5.0 | 99.58 ± 0.78 | 98.88 ± 0.68 | 1.28 | 1.32 |
Glynaseb | 5.0 | 100.2 ± 0.57 | 99.94 ± 0.97 | 0.53 | 2.9 |
aMarketed by Micro Labs Limited, Hosur, India.
bMarketed by USV Limited, Aurangabad, India.
cMean value of five determinations. Tabulated
The proposed method was successfully applied to the determination of GPZ in spiked urine sample with mean percentage recovery in the range of 103.1–104.5% as shown in Table
Results of determination of GPZ in spiked urine sample.
Spiked concentration µg mL−1 | Founda ± SD | % Recovery ± RSDb |
---|---|---|
150.23 | 155.46 ± 3.16 | 103.48 ± 1.01 |
200.22 | 209.24 ± 1.28 | 104.51 ± 1.02 |
250.32 | 258.12 ± 2.45 | 103.12 ± 0.98 |
aMean value of five determinations.
bRSD is relative standard deviation.
Results of recovery study by standard addition method.
Tablet studied | GPZ µg mL−1, tablet | GPZ µg mL−1, pure | Total GPZ found, µg mL−1 | Percent recovery of pure GPZ, |
---|---|---|---|---|
Dibizide | 49.21 | 50 | 100.23 | 101.03 ± 0.36 |
49.21 | 150 | 199.93 | 100.36 ± 0.45 | |
49.21 | 250 | 301.87 | 100.89 ± 0.89 | |
| ||||
Glynase | 48.67 | 50 | 99.89 | 101.24 ± 1.12 |
48.67 | 150 | 201.23 | 101.29 ± 0.1.02 | |
48.67 | 250 | 302.23 | 101.19 ± 0.98 |
The degradation was performed as per the current practice of ICH guidelines [
Chromatograms obtained for forced degradation.
5% H2O2 degradation
0.1N HCl degradation
0.1N NaOH degradation
H2O degradation
Thermal degradation
Photolytic degradation
A gradient RP-UPLC method was successfully developed for the estimation of PGZ in pharmaceutical dosage form and urine. The QbD project aims to encourage debate about quality in the complete development of the drug in a systematic manner. From a solid-state point of view, QbD implementation involves the design of manufacturing processes based on a thorough scientific understanding of the properties and stability of the components of the drug at critical points throughout the development. The experimental design describes the scouting of the key UPLC method components including column, temperature, pH, and mobile phase. The interrelationships are studied and the preliminary optimized conditions are obtained for each combination. Moreover, this approach ensures better design of products with fewer problems in development, reduces the number of trials required for postmarket changes, relies more on process and understanding and mitigation of risk, allows implementation of new technology to improve manufacturing without regulatory scrutiny, and enables possible reduction in overall costs of manufacturing resulting in less waste. The validated method is specific, linear, precise, accurate, robust, rugged, and stable for 24 hours and can be applied for the determination in formulated form. The drug is stable in acidic, thermal, photolytic, and hydrolytic conditions and degrades in basic, oxidative conditions. The potential of this QbD approach lies for the simultaneous determination of multiple forms of GPZ like pure drug, dosage forms, and in urine, and thus it should be implemented.
The authors thank Bal Pharma, Bangalore, India, for gifting pure glipizide. They are also thankful to the University of Mysore for providing the permission and facilities to do research work. Two of the authors (Cijo M. Xavier and K. B. Vinay) are grateful to Jubilant Life Sciences, Nanjangud, Mysore, India, for permission to pursue Ph.D. degree programme.