Chapter 2. Literature Review

Transcription

1 Chapter 2 Literature Review

2 Chapter 2: Literature Review Table 2.1: Official methods for estimation of anti-diabetics Sr. Drug/Sample and Method 1. Gliclazide (IP) Non-aqueous titration (Potentiometry) 2. Gliclazide (BP) Non-aqueous titration (Potentiometry) 3. Gliclazide tablets (IP) Liquid Chromatography 4. Glimepiride (IP) Liquid Chromatography 5. Glimepiride (BP) Liquid Chromatography Description Non-aqueous titration using 0.1 M perchloric acid as titrant and end-point is determined potentiometrically. Non-aqueous titration using 0.1 M perchloric acid as titrant and end-point is determined potentiometrically. Solvent mixture: ACN: Water (40: 60) Column: A stainless steel column 25 cm 4 mm, packed with endcapped octylsilane bonded to porous silica (4 µm) Mobile phase: TEA: Trifluoroacetic Acid: ACN: Water (0.1: 0.1: 45: 55, v/v/v/v) Flow Rate: 0.9 ml/min λ max: 235 nm Injection volume: 20 µl Column: A stainless steel column 25 cm 4.0 mm, packed with end capped octylsilane bonded to porous silica (4 µm) Mobile phase: Phosphate buffer (ph: 2.5): ACN (50:50, v/v) Flow Rate: 1 ml/min λ max: 228 nm Injection volume: 20 µl Column: 0.15 m 4.0 mm, end capped octadecylsilyl silica gel for chromatography R (4 µm) Mobile phase: Phosphate buffer (ph: 2.5): Ref K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 21

3 ACN (50:50, v/v) Flow Rate: 1 ml/min λ max: 228 nm Injection volume: 20 µl 6. Glimepiride (USP) Liquid Chromatography 7. Glimepiride tablets (IP) Liquid Chromatography 8. Glipizide (IP) Titration 9. Glipizide (BP) Titration Column: 4 mm x 25 cm column that contains packing L 1 Mobile phase: Phosphate buffer (ph: 2.1 to 2.7): ACN (50:50, v/v) Diluent: ACN: Water (4:1) Flow Rate: 1 ml/min λ max: 228 nm Injection volume: 20 µl Solvent mixture: ACN: Water (90: 10) Column: A stainless steel column 12.5 cm 4 mm, packed with octadecylsilane bonded to porous silica (4 µm) Mobile phase: Phosphate buffer (ph: 2.1): ACN (50:50, v/v) Flow Rate: 1 ml/min λ max: 228 nm Injection volume: 10 µl Dissolve accurately about 0.4 gm in 50 ml of dimethylformamide, add 0.2 ml of quinaldine red solution. Titrate with 0.1 M lithium methoxide until the colour changes from red to colourless. Dissolve 0.4 gm in 50 ml of dimethylformamide R. Add 0.2 ml of quinaldine red solution R. Titrate with 0.1 M lithium methoxide until the colour changes from red to colourless K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 22

4 10. Glipizide (USP) Liquid Chromatography 11. Glipizide tablets (IP) UV Spectrophotometry 12. Glipizide tablets (USP) Liquid Chromatography 13. Metformin Hydrochloride (IP) Non-aqueous titration (Potentiometry) 14. Metformin Hydrochloride (BP) Non-aqueous titration (Potentiometry) 15. Metformin Hydrochloride (USP) Non-aqueous titration (Potentiometry) 16. Metformin Hydrochloride tablets (IP) UV Spectrophotometry Column: 15 cm x 3.9 cm column that contains 5 µm packing L 1 Mobile phase: Phosphate buffer (ph: 6.00 ± 0.05): Methanol (55:45, v/v) Flow Rate: 1 ml/min λ max: 225 nm Injection volume: 20 µl Dissolve in methanol and measure the absorbance of solution at 274 nm. Column: 15 cm x 3.9 cm column that contains 5 µm packing L 1 Mobile phase: Phosphate buffer (ph: 6.00 ± 0.05): Methanol (55:45, v/v) Flow Rate: 1 ml/min λ max: 225 nm Injection volume: 20 µl Non-aqueous titration using 0.1 M perchloric acid as titrant and end-point is determined potentiometrically. Non-aqueous titration using 0.1 M perchloric acid as titrant and end-point is determined potentiometrically. Non-aqueous titration using 0.1 M perchloric acid as titrant and end-point is determined potentiometrically. Dissolve in water and measure the absorbance of solution at 232 nm K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 23

5 17. Metformin Hydrochloride tablets (USP) UV Spectrophotometry 18. Metformin Hydrochloride Sustained-release Tablets (IP) UV Spectrophotometry 19. Pioglitazone Hydrochloride (IP) Liquid Chromatography 20. Pioglitazone Tablets (IP) Liquid Chromatography 21. Rosiglitazone Maleate (IP) Liquid Chromatography Dissolve in water and measure the absorbance of solution at 232 nm. Dissolve in water and measure the absorbance of solution at 232 nm. Column: A stainless steel column 25 cm 4.6 mm, packed with octadecylsilane bonded to porous silica (5 µm) Column temperature: 25 C Mobile phase: 0.01 M potassium dihydrogen phosphate: ACN (50: 50, v/v) Flow Rate: 1 ml/min λ max: 225 nm Injection volume: 20 µl Solvent mixture: Water: ACN (35: 65) Column: A stainless steel column 25 cm 4.6 mm, packed with octadecylsilane bonded to porous silica (5 µm) Mobile phase: Phosphate buffer (ph: 5): ACN (35:65, v/v) Flow Rate: 1.5 ml/min λ max: 270 nm Injection volume: 20 µl Column: A stainless steel column 25 cm 4.6 mm, packed with octadecylsilane bonded to porous silica (5 µm) Mobile phase: Phosphate buffer (ph: 3): ACN: Methanol (65:25:10, v/v/v) Flow Rate: 1 ml/min K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 24

6 λ max: 235 nm Injection volume: 10 µl 22. Rosiglitazone Tablets (IP) Liquid Chromatography Column: A stainless steel column 25 cm 4.6 mm, packed with octadecylsilane bonded to porous silica (5 µm) Mobile phase: Phosphate buffer (ph: 3): ACN: Methanol (65:25:10, v/v/v) Flow Rate: 1 ml/min λ max: 235 nm Injection volume: 10 µl 71 Table 2.2: Reported methods for estimation of Gliclazide Sr. Drug/Sample Method Description 1. Gliclazide in Liquid Column: Chromolith human plasma Chromatography Performance RP-18 (100 mm 4.6 mm) Mobile phase: 0.01 M disodium hydrogen phosphate buffer (ph: 4.0): ACN (52:48, v/v) λ max: 230 nm 2. Gliclazide in HPLC with Column: C 18 (150 mm 4.6 plasma electrochemical mm) detection Mobile phase: 70 mm disodium tetraborate, ph 7.5, containing 26.5% of ACN 3. Gliclazide; Stability-Indicating Column: C 18 Gliclazide HPLC-UV Method Mobile phase: ACN: tablets ammonium acetate solution (0.025 M, ph 3.5) (40: 60, v/v) Ref K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 25

7 Flow Rate: 0.25 ml/min λ max: 235 nm 4. Gliclazide Semi-micro HPLC Column: Semi-micro C Gliclazide in serum 6. Gliclazide in human serum 7. Gliclazide in human serum 8. Gliclazide in pharmaceutical Mobile phase: 40 mm KH 2 PO 4 (ph 4.6): ACN: Isopropyl alcohol (5:4:1, v/v/v) Flow Rate: 0.22 ml/min λ max: 229 nm HPLC using solidphase Column: Octadecyl silica (150 extraction mm x 4.6 mm, 5 µm) Mobile phase: 0.04 M potassium dihydrogen phosphate (ph 4.6): ACN: Isopropyl alcohol (5:4:1, v/v/v) λ max: 227 nm HPLC Column: C 8 Mobile phase: ACN: Water (45:55, v/v), ph 3 Retention time: 6.8 min λ max: 230 nm HPLC using an Column: Column packed with a anion-exchange macroporous anion-exchange resin resin, Diaion CDR-10 Mobile phase: ACN: Methyl alcohol: 1.2 M ammonium perchlorate (4:3:7, v/v/v) Flow Rate: 0.4 ml/min Retention time: 15 min λ max: 230 nm Spectrophotometry The proposed method is based upon the formation of ternary K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 26

8 and biological fluids 9. Gliclazide in plasma 10. Gliclazide in pharmaceutical preparations 11. Gliclazide in human serum complex between palladium (II), eosin and Gliclazide in the presence of methyl cellulose as a surfactant and acetate buffer of ph 4.5. Ternary complex showed absorption maximum at 550 nm. Radial compression Gliclazide and the internal Reversed-Phase standard, 3-chloroGliclazide, Liquid are eluted after 4.4 and 6.8 Chromatography min, respectively. LOD: 0.5 mg/l λ max: 229 nm Capillary Gas Column: 25 m x 0.25 mm id, Chromatography 0.2 µm film thickness CP-WAX 58 (FFAP)-CB WCOT fused silica Retention Time: 36 min Radioimmunoassay Antisera A and B against Gliclazide were obtained from guinea pigs immunized with conjugates A and B prepared by coupling Gliclazide homologues, 1-(p- toluenesulfonyl)-3-(4'- carboxypiperidino) urea and 1- (4-methyl-3- carboxybenzenesulfonyl)-3-(3- azabicyclo [3, 3, 0] oct-3-yl) urea, to bovine serum albumin. 3H-Gliclazide was used as a tracer K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 27

9 Dextran-coated charcoal was used to separate bound and free 3H-Gliclazide in the reaction mixture. 12. Gliclazide in human plasma 13. Gliclazide in plasma 14. Gliclazide in plasma 15. Gliclazide in plasma 16. Gliclazide in serum Liquid Chromatography - Mass Spectrometry Column: C 18 column interfaced with a triple quadrupole mass spectrometer Mobile phase: ACN: Water containing 10 mmol/l ammonium acetate, ph 3.5 adjusted with acetic acid (75:25, v/v) Flow Rate: 1.0 ml/min HPLC Plasma Gliclazide concentrations were determined using HPLC and pharmacokinetic parameters were analyzed by non compartmental analysis HPLC Plasma harvested from blood was analyzed for Gliclazide by validated HPLC method. RP HPLC Blood samples were collected upto 72 hrs after administrations and plasma samples assayed for Gliclazide concentrations using RP HPLC method with UV detection. HPLC Gliclazide in serum was determined by HPLC with flurescence detector K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 28

10 17. Gliclazide RIA UV HPLC 18. Gliclazide in HPLC human plasma Thromboxane B 2 (TXB 2 ) and betathromboglobulin (beta TG) were measured by radioimmunoassay. Lipid peroxides were measured asa total diene cojugates (DC) by scanning UV spectrophotometry and thiobarbituric acid reactivity (TBA) by absorbance at 532 nm. Oxidation products of proteins were represented by fluorescence/uv ratio of immunoglobulin G (*IgG) separated from serum by HPLC. Column: Hypersil ODS (250 mm x 4.6 mm, 5 µm) Mobile Phase: 0.02 M KH 2 PO 4 : Methanol (50:50, v/v) ph adjusted to 3 with H 3 PO 4 Amperometric detection: + 0.9V Table 2.3: Reported methods for estimation of Glimepiride Sr. Drug/Sample Method Description 1. Glimepiride in LC MS MS Column: ACE 5 C 18 (50 human plasma mm 4 mm, 5 μm) Column Temperature:30 C Mobile phase: 200 ml water, 450 ml ACN, 350 ml methanol, Ref. 90 K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 29

11 0.6 ml glacial acetic acid Flow Rate: 0.5 ml/min Internal Standard: Gliclazide Retention time: Glimepiride: 1.65 min Gliclazide: 1.36 min Run time: 2.5 min 2. Cis-Isomer of Glimepiride in a bulk drug substance 3. Glimepiride and its related impurities 4. Glimepiride in human plasma 5. Glimepiride in pharmaceutical formulations RP-LC Column: Waters Symmetry column (50 mm 4.6 mm, 3.5 μm) Mobile phase: Water: Tetrahydrofuran (75:25, v/v) LC Column: Phenomenex Luna C 8 (250 mm 4.6 mm, 5 μm) Column Temperature:35 C Mobile phase: Phosphate buffer (ph 7.0): ACN: Tetrahydrofuran (73:18:09, v/v/v) Flow Rate: 1 ml/min λ max: 228 nm LC-MS-MS Column: C 18 Column Temperature:35 C Mobile phase: ACN: Deionized water (adjusted to ph 3.5 with acetic acid) (80:20, v/v) Flow Rate: 200 μl/min HPLC and First- HPLC: Derivative Column: C 18 (250 mm 4.6 Spectrophotometry mm, 5 μm) Mobile phase: ACN and 2% K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 30

12 formic acid solution, ph 3.5 (80:20, v/v) λ max: 228 nm 1 st order derivative spectrophotometric method: Detect the complex at nm 6. Glimepiride Derivative UV Spectrophotometry 7. Glimepiride in Semi-microbore human plasma HPLC with columnswitching Second order derivative UV spectrophotometry: The second order derivative spectra was calculated using peak to peak (λ DMF = nm), peak to zero (λ DMF =268.2 nm) and tangent (λ DMF = nm) method. Column: Pre-separation: Capcell Pak MF Ph-1 (10 mm 4.0 mm) Intermediate separation: Capcell Pak C 18 UG 120 U (35 mm 2.0 mm) Final separation: Capcell Pak MG C 18 (250 mm 1.5 mm, 5 μm) Column Temperature:30 C Mobile phase: ACN: 10 mm potassium phosphate buffer containing 0.04% of TEA (52:48, v/v, ph 2.18) Washing Solvent: ACN: 10 mm K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 31

13 phosphate buffer containing 0.04% of TEA (ph 2.18) (20:80, v/v) Flow Rate: 0.1 ml/min λ max: 228 nm Retention time: 34.9 min 8. Glimepiride in human plasma Liquid Chromatography Column: XTerra C 18 (50 mm 2.1 mm, 5 μm) Electrospray Mobile phase: Ammonium Ionization Tandem acetate buffer (0.02 M, Mass Spectrometry ph=3.5): ACN: Methanol (40:35:25, v/v/v) Flow Rate: 0.28 ml/min 9. Glimepiride in Liquid-liquid Glimepiride and the internal human plasma extraction based on standard (IS) Glibenclamide 96-well format were extracted from human micro-tubes and plasma by LLE, using a mixture Liquid of ethyl acetate/diethyl ether Chromatography/ 50:50 (v/v) as the organic Tandem Mass solvent. The analyte and IS Spectrometry were dissolved in a small volume of a reconstitution solution, an aliquot of which was analyzed by reversedphase LC/MS/MS with positive ion electrospray ionization, using multiple reaction monitoring. 10. Glimepiride HPLC Column: C 18 Mobile phase: ACN: Phosphate buffer (ph 3.5, 0.03 M) (48:52, K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 32

14 v/v) The susceptibility of Glimepiride to hydrolytic decomposition increased in following manner: neutral condition < alkaline condition < acid condition < oxidative condition. 11. Glimepiride in human plasma Liquid Chromatographic Tandem Mass Spectrometry Column: Hypersil ODS (250 mm x 4.6 mm, 5 µm) Column Temperature:25 C Mobile phase: 0.05 M Formic acid: ACN (28:72, v/v) Flow Rate: 0.3 ml/min 12. Glimepiride and HPLC after precolumn The assay involves extraction its metabolites with diethyl ether, thermolysis in human derivatization of the sulphonylureas at 100 C serum and and trapping of the resulting urine amines with 2,4- dinitrofluorobenzene. The derivatives were quantitated on a reversed-phase column by absorbance at 350 nm using a step gradient for the three compounds in serum and an isocratic run for the metabolites in urine. 13. Glimepiride RP HPLC Column: Hypersil C 18 (15 cm x 3.9 mm) Mobile phase: ACN: 0.05 M monobasic potassium K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 33

15 phosphate (ph 6.0) (40:60, v/v) Flow Rate: 1.5 ml/min λ max: 210 nm Retention Time: 7.8 min 14. Degradation products of Glimepiride 15. Glimepiride in rat serum LC UV PDA and LC Glimepiride was subjected to MS forced decomposition under the conditions of hydrolysis, oxidation, dry heat and photolysis, in accordance with the ICH guideline Q1A (R2). Column: Spherisorb C 8 (150 mm 4.6 mm, 5 µm) Mobile phase: ACN: Ammonium acetate (ph: 3.0; 0.02 M) (20:80, v/v) Flow Rate: 1.5 ml/min Injection Volume: 20 µl λ max: 235 nm RP HPLC Column: C 18 Mobile phase: Methanol: 10 mm phosphate buffer (80:20, v/v) adjusted to ph 3.0 with OPA Flow Rate: 1.0 ml/min λ max: 230 nm Internal Standard: Gliclazide Retention time: Glimepiride: 5.5 min Gliclazide: 4.0 min Run time: 10 min K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 34

16 16. Glimepiride in Liquid Column: YMC Propack C 18 ( human plasma Chromatography- mm x 4.6 mm) Electrospray Mobile phase: Ammonium Ionization Tandem acetate buffer: ACN : Methanol Mass Spectrometry (30:60:10, v/v/v) Flow Rate: 0.5 ml/min Table 2.4: Reported methods for estimation of Glipizide Sr. Drug/Sample Method Description 1. Glipizide RP HPLC Column: Reversed-phase C 18 Mobile phase: ACN: 0.05 M KH 2 PO 4 buffer adjusted to ph 3.5 with OPA λ max: 275 nm 2. Glipizide RP HPLC Column: BDS Hypersil C 18 (250 mm X 4.6 mm, 5 µm) Mobile phase: 20 mm Ammonium acetate (ph 4.5): ACN (55: 45, v/v) Flow Rate: 1 ml/min λ max: 230 nm Retention time: 8.2 min 3. Glipizide in RP HPLC Column: Spherisorb ODS human plasma Mobile phase: ACN: 0.01 M and urine phosphate buffer ph 3.5 (35:65, v/v) Injection volume: 20 µl λ max: 275 nm 4. Glipizide LC and LC-MS Forced degradation studies on Glipizide are conducted under Ref K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 35

17 the conditions of hydrolysis, oxidation, photolysis, and dry heat. The solutions are subjected to liquid chromatographic (LC) investigations to establish the number of products formed in each condition. The degradation products are characterized through isolation and subsequent NMR, IR, and MS spectral analyses, or through LC-MS fragmentation pattern study. 5. Glipizide in bulk Stripping The electrochemical behavior 110 form and Voltammetric of Glipizide at the hanging pharmaceutical mercury drop electrode formulation (HMDE) was studied in B-R universal buffers of ph Table 2.5: Reported methods for estimation of Metformin Sr. Drug/Sample Method Description 1. Metformin in HPLC-UV Column: Reversed-phase human plasma phenyl column Column Temperature: 40 C Mobile phase: Phosphate buffer and ACN Flow Rate: 1.0 ml/min λ max : 236nm Ref. 111 K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 36

18 2. Metformin HCl HPTLC Mobile phase: Methanol: Chloroform: Ammonium acetate (6:3:1, v/v/v) Densitometry at 236 nm 3. Metformin Dried Blood Spot The method is based on RP Liquid HPLC with ultraviolet detection Chromatography 4. Metformin in HPLC Column: Silica (250 mm 4.6 human plasma mm, 5 μm) Mobile phase: ACN: 40 mm aqueous sodium dihydrogen phosphate (25:75, v/v), ph 6 5. Metformin in LC/MS/MS Column: Capcell Pak SCX human plasma column with a normal-phase gradient system followed by semi-micro LC/MS/MS in positive ion selected reaction monitoring mode using electrospray ionization Run time: 7min/sample 6. Metformin in HPLC-Tandem Mass Column: C 18 (100 mm 2.0 mm, human plasma Spectrometry 3 μm) connected to a guard column MS-pursuit (0.20 mm 0.20 mm, 5 μm) Mobile phase: Water: ACN: Formic acid (55: 45: 0.048, v/v/v) Run time: 3 min Detection: Mass spectrometer Internal Standard: Metoprolol K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 37

19 7. Metformin in plasma LC/MS/MS Two Methods: 1) First method: Column: YMC cyano (2 mm x 50 mm, 3 µm) Mobile phase: 100% ACN to ACN: Water (80:20, v/v) containing 10 mm ammonium acetate and 1% acetic acid was applied Run time: 2 min 2) Second method: Column: YMC cyano (2 mm x 10 mm, 5 µm) Mobile phase: ACN: Methanol (95:5, v/v) Run time: 1 min 8. Metformin HPLC Column: ODS (250 mm 4.6 hydrochloriderelated mm, 5 µm) Mobile phase: Methyl alcohol substances (1 mmol/l): Sodium dodecylsulfate in (10 mmol/l) phosphate salt solution (60:40) (ph=5.5) Flow Rate: 0.8 ml/ min λ max: 232 nm 9. Metformin Voltammetry Molecular wires containing copper (II) (CuMW), in the form of the coordination polymer (Cu (II) 4(bpp) 4 (maa) 8 (H 2 O) 2). 2H 2 O (bpp=1, 3-bis (4-pyridyl) propane, maa=2-methylacrylic K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 38

20 acid), and multiwalled carbon nanotubes (CNT) have been combined to prepare a paste electrode (CuMW/CNT/PE). 10. Metformin in urine 11. Metformin in human plasma 12. Metformin in human plasma 13. Metformin in human plasma and breast milk Square-wave The electrode reaction was Voltammetry analyzed under conditions of linear sweep voltammetry (LSV), differential pulse voltammetry (DPV) and Osteryoung-type square-wave voltammetry (SWV). HPLC Mobile phase: 0.01 M potassium dihydrogen orthophosphate (ph 3.5): ACN (60:40, v/v) Flow Rate: 1.0 ml/min λ max: 234 nm HPLC Column: Silica Mobile phase: ACN (250 ml) in ph 7, 0.03 M diammonium hydrogen phosphate buffer (750 ml) Flow Rate: 1 ml/min λ max: 240 nm HPLC After proteins were precipitated with ACN, Metformin and the internal standard buformin were resolved on a cation-exchange column and detected by UV detection at 236 nm K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 39

21 14. Metformin in human plasma Liquid Chromatography- Tandem Mass Spectrometry Column: Nucleosil C 18 (50 mm x 4.6 mm, 5 µm) Mobile phase: ACN: Methanol: 10 mm ammonium acetate ph 7.0 (20:20:60, v/v/v) Flow Rate: 0.65 ml/min 15. Metformin HPLC after precolumn Column: Lichrosper C 18 (6.0 mm Hydrochloride x 150 mm, 5 µm) derivatization Mobile phase: Methanol: Water (70:30, v/v) λ max: 256 nm 16. Metformin in HPLC Column: Cation-exchange plasma extraction column (7.5 mm x 4.6 mm), a column switching valve, and a cation-exchange analytical column (250 mm x 4.6 mm) λ max: 232 nm 17. Metformin RP-HPLC Column: C 18 Mobile phase: Methanol: Water (30:70, v/v) Flow Rate: 0.5 ml/min Retention time: 4.4 min λ max: 233 nm Internal standard: Diazepam 18. Metformin in HPLC Column: Packed with plasma microbondapak phenyl λ max: 236 nm Flow Rate: 1.35 ml/min 19. Metformin in Liquid- The assay requires only 0.5 ml plasma and Chromatography of sample and involves K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 40

22 urine pretreatment with trichloroacetic acid containing an internal standard (1- propylbiguanide), followed by centrifugation and injection into the chromatograph. The column eluent is monitored at 230 nm. 20. Metformin in Liquid Column: C human plasma Chromatography- Detection: Tandem mass Tandem Mass spectrometry Spectrometry Run time: 3.4 min 21. Metformin Capillary Fused silica capillary (60 cm x 131 dosage Electrophoresis 50 µm), Phosphate buffer (50 formulations mm, 3.0 ph): ACN (95:5, v/v) as background electrolyte (BGE), 20 kv applied voltage with UV detection at 254 nm and at a working temperature of 23 ±1 C 22. Metformin in HPLC The method involved direct 132 antemortem injection of a protein-free serum and filtrate into the liquid postmortem chromatograph. specimens 23. Metformin in Liquid HO*-induced oxidation of 133 aqueous Chromatographic/ Metformin was studied in solutions Electrospray aqueous solution, in both Ionization Mass aerated and deaerated Spectrometry conditions. Gamma radiolysis of water was used to generate HO* free radicals, capable of K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 41

23 initiating one-electron oxidation of Metformin. Oxidation end-products were identified by direct infusion MS and HPLC/MS. 24. Metformin Chemiluminescence This method is based on hydroxyl radical chemiluminescence. The hydroxyl radical generated by reaction of Cu(II) and hydrogen peroxide oxidizes rhodamine B (RhB) to produce weak CL which can be enhanced by Metformin. 25. Metformin in Fourier Continuous An easy and fast Fourier its Cyclic Voltametric transform continuous cyclic pharmaceutical voltametric technique for formulation monitoring of ultra trace amounts of Metformin in a flow-injection system has been introduced in this work. 26. Metformin Chemiluminometric The developed methodology was based on the Metformininduced inhibition (Metformin acts as a Cu(II) scavenger) of the catalytic effect of Cu(II) ions on the chemiluminescent reaction between luminol and hydrogen peroxide. 27. Metformin Liquid One developed and validated in Chromatography 1990 and one developed and K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 42

24 validated in 1996, are compared. The first method, using an octadecyl phase and an ion pairing agent in the eluent, could not be reproduced some five years later, but another method, using a phenyl phase and no ion pairing agent, could be successfully applied. 28. Microassay of plasma and erythrocyte Metformin 29. Metformin in plasma 30. Metformin in human plasma HPLC The proposed HPLC method allows the quantification of plasma and intra-erythrocyte Metformin after simple acid deproteinisation of blood samples and injection on a cation exchange column. Cation-Exchange Column: Cation-exchange HPLC Mobile phase: Potassium dihydrogen phosphate buffercontaining ACN λ max: 236 nm HPLC Column: Mubondapak C 18 (150 mm x 4.6 mm) Mobile phase: 40% ACN: 0.01 M sodium dodecyl sulphate: 0.01 M sodium dihydrogen phosphate, and distilled water to 100%, adjusted to ph 5.1 λ max: 235 nm Flow Rate: 1.5 ml/min K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 43

25 31. Metformin in Laser Diode Thermal A simple, rapid and robust high- 141 mouse, rat, dog Desorption/Atmosp throughput assay for the and human heric Pressure quantitative analysis of plasma Chemical Ionization Metformin in plasma from samples Tandem Mass different species using LDTD- Spectrometry APCI-MSMS was developed for (LDTD-APCI-MSMS) use in a Pharmaceutical discovery environment. In order to minimize sample preparation a generic protein precipitation method was used to extract Metformin from the plasma. Sr. Table 2.6: Reported methods for estimation of Pioglitazone Drug/Sample Method Description Ref. 1. Pioglitazone in human plasma HPLC Column: Apollo C 18 Mobile phase: Methanol: ACN: Mixed phosphate buffer (ph 2.6; 10mM) (40:12:48, v/v/v) Flow Rate: 1.2 ml/min Linearity: ng/ml (r 2 = ) λ max: 269 nm Pioglitazone Liquid The method involved a solid 143 and its Chromatography and phase extraction (SPE) of metabolites in Solid Phase Pioglitazone, its metabolites, human serum Extraction and the internal standard (U ) from serum using C 18 SPE columns with an elution solvent of 0.5 ml of ACN: water K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 44

26 (35:65, v/v) Column: Zorbax RX-C 8 (250 mm 4.6 mm, 5 μm) Mobile phase: ACN: Water (40:60, v/v) containing 3 ml acetic acid per liter mobile phase (apparent ph 5.5) Flow Rate: 0.5 ml/min λ max: 269 nm Linearity: µg/ml 3. Pioglitazone HPLC The method for serum involved 144 and its the solid-phase and liquid- metabolites in liquid extraction. Urine with human serum and without enzymatic and urine hydrolysis using β- glucuronidase was treated with liquid-liquid extraction. The compounds in the extract were analyzed using HPLC with UV detection at 269 nm. 4. Pioglitazone in Direct-injection Column: Luna C 18 (4.6 mm 145 human serum High-Performance 50 mm, 5 μm) Liquid Mobile phase: 5 mm Chromatography ammonium acetate and ACN Mass Spectrometry Flow Rate: 1.35 ml/min Linearity: ng/ml 5. Pioglitazone LC MS/MS The compounds were eluted 146 and its two isocratically on a C-18 column, active ionized using a positive ion metabolites in atmospheric pressure human plasma electrospray ionization source K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 45

27 and analyzed using multiple reactions monitoring mode. Run Time: 2.5 min Retention times: PIO: 1.45 min M-III: 1.02 min M-IV: 0.95 min Linearity: ng/ml (r 2 > ) 6. Pioglitazone HPLC and MEKC MEKC method: 147 Hydrochloride Pioglitazone and its in bulk and unsaturated impurity were pharmaceutical separated by MEKC in less than formulations 7 min using a 43 cm 50 μm i.d. uncoated fused-silica capillary with extended light path for better sensitivity (25 kv at 30 C) and a background electrolyte (BGE) consisting of 20% ACN (v/v) in 20 mm sodium borate buffer ph 9.3 containing 50 mm sodium dodecyl sulphate (SDS) HPLC method: Column: C 18 (250 mm 4.6 mm, 5 μm) Mobile phase: ACN: 10 mm potassium dihydrogen phosphate buffer (50:50, v/v), adjusting the ph to 6.0 with 0.1 M KOH K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 46

28 7. Pioglitazone Stability indicating Column: Hypersil C 8 (250 mm 148 from tablets RP-HPLC 4.6 mm) Mobile phase: ACN: 0.15% v/v TEA (40:60, v/v), adjusted to ph 4.6 with OPA Flow Rate: 1.5 ml/min λ max: 220 nm Retention Time: 7.6 min 8. Pioglitazone in human plasma HPLC Column: Nova-Pak C 8 Mobile phase: ACN: 140mM K 2 HPO 4 (40:60, v/v, ph: 4.45) Flow Rate: 1.4 ml/min λ max: 269 nm Run Time: 7 min Linearity: ng/ml (r 2 > 0.999) Internal Standard: Ethyl Paraben Pioglitazone in Solid-Phase Column: Octadecylsilane 150 dog serum Extraction and HPLC Mobile phase: ACN: Water (41:59, v/v) containing 1.2 ml/l acetic acid (ph 6.0 ± 0.05) Flow Rate: 1.4 ml/min λ max: 229 nm Linearity: µg/ml 10. Pioglitazone Hydrochloride in tablets HPLC Column: Symmetry C 18 (250 mm 4.6 mm, 5 μm) Mobile phase: Mixture of ammonium formate buffer adjusted with formic acid to ph 4.1 and ACN 151 K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 47

29 11. Pioglitazone Hydrochloride from tablets RP-HPLC Column: Hypersil C 8 Mobile phase: ACN: Water(60:40, v/v) adjusted to ph 6.0 with 0.1 % v/v glacial acetic acid Flow Rate: 1 ml/min λ max: 266 nm Retention Time: 6.40 min Pioglitazone RP-HPLC Column: Nova-Pak C 18 (3.9 mm 153 Hydrochloride x 150 mm, 5 µm) in tablets Mobile phase: Ammonium formate buffer adjusted with formic acid to ph 3: ACN (75:25, v/v) Flow Rate: 1 ml/min λ max: 225 nm Run Time: 12 min 13. Pioglitazone HCl in pharmaceutical dosage forms RP-HPLC Column: C 18 (250 mm 4.6 mm, 5 μm) Mobile phase: Acetate buffer: ACN (55:45, v/v) Flow Rate: 1 ml/min λ max: 254 nm Retention Time: min 154 Sr. Table 2.7: Reported methods for estimation of Rosiglitazone Drug/Sample Method Description Ref. 1. Rosiglitazone HPLC The extraction from plasma 155 in Plasma was performed using solidphase extraction (SPE) on C4 K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 48

30 silica (100 mg) disposable extraction cartridges (DEC). Column: Phenomenex Synergi MAX-RP (150 mm 4.6 mm, 4 µm) Mobile phase: 0.01 M ammonium acetate, ph ACN (65:35, v/v) Detection: Fluorescence Internal Standard: (3S)-3-OHquinidine Linearity: ng/ml (r 2 =0.9959) 2. Rosiglitazone Solid-Phase Rosiglitazone was extracted 156 in urine Extraction coupled from urine using a SPE with Liquid cartridge of 50 mg C 8 sorbent Chromatography and ACN used as the eluting Tandem Mass solvent. Spectrometry Column: RP Rosiglitazone Gradient Liquid Column: Phenyl (250 mm and Metformin Chromatography mm, 5 μm) in plasma Mobile phase: ACN: 5 mm acetate buffer ph 5.5 (75:25, v/v) Flow Rate: 1.0 ml/min λ max: 245 nm Run time: 10 min 4. Rosiglitazone HPLC Column: Novapak C 18 (100 mm 158 in human 5 mm, 4 μm) protected by a plasma Guard-Pak C 18 cartridge Mobile phase: 0.01 M K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 49

31 Ammonium acetate (ph 8): ACN (65:35, v/v) Fluorescence Detection: λ exi: 247 nm λ emi: 367 nm 5. Rosiglitazone Liquid Column: pro C in human Chromatography Mobile phase: Ammonium plasma Mass Spectrometry acetate buffer (0.02 M, ph 6.5): ACN (47:53, v/v) Linearity: ng/ml 6. Rosiglitazone Voltammetry The voltammetric behavior of 160 in Rosiglitazone was studied using Pharmaceutical direct current (DC t ), differential Preparations pulse (DPP), and alternating and Human current (AC t ) polarography. Plasma The drug manifests cathodic waves over a ph range of In Britton-Robinson buffer (BRb; ph 4), the diffusion current-concentration relationship was found to be rectilinear over a range of 4-24 µg/ml and µg/ml using DC t and DPP modes, respectively. 7. Rosiglitazone First-Derivative First-derivative 161 in coated Spectrophotometry spectrophotometry, applying tablets the peak-zero method, was developed for the determination of Rosiglitazone in coated tablets. The solutions K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 50

32 of standard and sample were prepared in ethanol. 8. Rosiglitazone in human plasma 9. Rosiglitazone in human plasma HPLC Column: pro C 18 Mobile phase: ACN: 20 mm ammonium acetate ph 7.5 (52: 48, v/v) Flow Rate: 0.2 ml/min Fluorescence Detection: λ exi: 247 nm λ emi: 367 nm HPLC Column: Hichrom KR 100 C 18 ( 250 mm x 4.6 mm) Mobile phase: Potassium dihydrogen phosphate buffer (0.01 m, ph 6.5): ACN: Methanol (40:50:10, v/v/v) Flow Rate: 1.0 ml/min Fluorescence Detection: λ exi: 247 nm λ emi: 367 nm Internal Standard: Celecoxib Linearity: ng/ml Rosiglitazone Liquid Column: Zorbax SB C 18, 15-cm 164 in human Chromatography Mobile phase: Methanol and a plasma mixed phosphate buffer (10 mm monobasic sodium phosphate and dibasic sodium phosphate, ph adjusted to 2.6 with OPA) (30:70, v/v) λ max: 245 nm Linearity: ng/ml K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 51

33 11. Rosiglitazone Liquid Column: Alltima phenyl ( in human Chromatography mm x 4.6 mm, 5 µm) plasma Mobile phase: 10 mm sodium acetate: ACN (ph 5; 60:40, v/v) Flow Rate: 1.0 ml/min Detection: Fluorescence at (EX/EM) 247/367 for Rosiglitazone and 235/310 for the internal standard Betaxolol 12. Rosiglitazone in human plasma 13. Rosiglitazone, cilostazol, and 3,4-dehydrocilostazol in rat plasma HPLC Column: Alltima phenyl (250 mm x 4.6 mm, 5 µm) Mobile phase: Phosphate buffer, ACN and methanol Flow Rate: 1.0 ml/min Fluorescence Detection: λ exi: 247 nm λ emi: 367 nm HPLC Column: C 18 Mobile phase: ACN: Potassium di-hydrogen phosphate buffer (35:65, v/v) Flow Rate: 1.2 ml/min Detection: UV at dual wavelength of 226 nm (RSG and 3, 4-dehydro-cilostazol) and 257 nm (Cilostazol) Rosiglitazone Liquid Column: Symmetry C in bulk and Chromatography Mobile phase: Phosphate pharmaceutical buffer (ph:6.2):acn(50:50, v/v) formulation Flow Rate: 1.0 ml/min λ max: 245 nm K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 52

34 Table 2.8: Reported methods for estimation of Sitagliptin Sr. Drug/Sample Method Description 1. Sitagliptin in First order The λmax of Sitagliptin in bulk and in derivative UV- methanol and water was found Formulation Spectrophotometry to be 267 nm. The same spectrum was derivatised into first order derivative; showed maximum amplitude of the trough at 275 nm. Linearity: μg/ml (r = 0.998) 2. Sitagliptin in Turbulent flow A narrow bore large particle human urine online extraction size reversed-phase column and and tandem mass (Cyclone, 50 mm 1.0 mm, 60 hemodialysate spectrometry µm) and a BDS Hypersil C 18 column (30 mm 2.1 mm, 3 µm) were used as extraction and analytical columns, respectively. Linearity: µg/ml 3. Sitagliptin Stability Indicating The UV spectrum was scanned Phosphate in UV between nm and 267 Bulk and Tablet Spectrophotometry nm was selected as maximum Dosage Form wavelength for absorption. Linearity: mg/ml Forced degradation studies include the effect of temperature, oxidation, photolysis and susceptibility to hydrolysis across a wide range Ref K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 53

35 of ph values, were carried out according to ICH requirements. 4. Sitagliptin Phosphate 5. Sitagliptin phosphate 6. Sitagliptin in its pharmaceutical preparations First Order In distilled water λmax of Derivative UV Sitagliptin Phosphate was Spectrophotometry found to be nm. The same spectra was derivatized using derivative mode into first order derivative at ΔN =5, the amplitude of peak was measured at nm. Linearity: 5 35 µg/ml RP-HPLC Column: Symmetry C 18 (150 mm 4.6 mm, 5 μm) Mobile Phase: Methanol: 0.1% perchloric acid solution (32 68, v/v) Flow Rate: 1.0 ml/min λmax: 268 nm Linearity: μg/ml (r=0.9996) Spectrophotometry The proposed method is based on condensation of the primary amino group of Sitagliptin phosphate with acetyl acetone and formaldehyde producing a yellow colored product, which is measured spectrophotometrically at 430 nm. The color was stable for about 1 hour. Linearity: 5 25 μg/ml K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 54

36 7. Sitaglitpin in human plasma 8. Sitagliptin phosphate 9. Sitagliptin Phosphate in API and its unit dosage forms Liquid Following liquid-liquid Chromatography Tandem Mass Spectrometry extraction, the analytes were separated using an isocratic mobile phase on reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode. Linearity: ng/ml RP-HPLC Column: Phenomenex C 18 (250 mm 4.6mm, 5 μm) Mobile Phase: 0.5% v/v of TEA solution: ACN (77:23, v/v). The ph of 0.5% v/v TEA was adjusted to 6.8 using OPA Flow Rate: 1.0 ml/min λmax: 267 nm Retention time: Sitagliptin: 6.1 min Internal standard: 7.7 min Linearity: ng/ml Spectrophotometry Two simple, accurate, sensitive and reproducible visible spectrophotometric methods (A & B) have been developed for the determination of Sitagliptin Phosphate (SGP) in bulk and also in pharmaceutical formulations. The proposed methods are based on complexation of the drug with Bromo Thymol Blue (BTB K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 55

37 Method A) & Bromo Cresol Green (BCG-Method B), extracted with chloroform, showing absorbance maxima at 412 nm and 419 nm, respectively. Linearity: Method A: μg/ml Method B: μg/ml Table 2.9: Reported methods for estimation of Vildagliptin Sr. Drug/Sample Method Description 1. Vildagliptin in Liquid Vildagliptin (VDG) was the Presence of Chromatography determined in the presence of its synthetic 3-amino-1-adamantanol (AAD), intermediate a synthetic intermediate and impurity of VDG. Ref. 178 Column: Symmetry Waters C 18 (150 mm 4.6 mm, 5 μm) Mobile phase: Potassium dihydrogen phosphate buffer ph (4.6): ACN: Methanol (30:50:20, v/v/v) Flow Rate: 1 ml/min λ max: 220 nm Retention time: 10 mins Linearity: μg/ml 2. Sitagliptin and Spectrophotometry The proposed methods are 179 Vildagliptin in based on the charge transfer bulk and dosage complexes of Sitagliptin K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 56

38 forms phosphate and Vildagliptin with 2, 3-dichloro-5, 6-dicyano-1, 4- benzoquinone (DDQ), 7, 7, 8, 8- tetracyanoquinodimethane (TC NQ) and tetrachloro-1, 4- benzoquinone (p-chloranil). Linearity: Sitagliptin: μg/ml, μg/ml and μg/ml with DDQ, TCNQ and p- chloranil, respectively. Vildagliptin: μg/ml, μg/ml and μg/ml with DDQ, TCNQ and p- chloranil, respectively. Table 2.10: Reported methods for estimation of Metformin and Gliclazide Sr. Drug/Sample Method Description 1. Gliclazide and Spectrophotometry Three Methods: Metformin 1) First method is based on an Hydrochloride equation of area calculation of curve at two wavelength regions (228.6 to 224 nm and 235 to 231 nm) 2) Second method employs simultaneous equation at two wavelengths corresponding to and nm 3) Third method employs Ref. 180 K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 57

39 derivative spectroscopy at zero crossing points (226 and nm) in first order derivative spectra 2. Metformin and Gliclazide in tablets 3. Metformin and Gliclazide in human plasma 4. Gliclazide and Metformine Hydrochloride in tablet dosage form RP HPLC Column: Shimpack CLC C 8 (250 mm x 4 mm, 5 µm) Mobile phase: M disodium hydrogen phosphate: ACN (25:75, v/v) with ph adjusted to 3.2 with dilute OPA Flow Rate: 1 ml/min Retention time: Metformin: 2.8 min Gliclazide: 4.3 min λ max: 240 nm Liquid LC/MS/MS analysis using Chromatography- electro-spray ionization (ESI). Tandem Mass Column: Hypersil BDS C 18 (50 Spectrometry mm x 2.1 mm, 3 µm) Run time: 2.0 min UV Two Methods: Spectrophotometry 1) First method based on solving of simultaneous equation using 228 nm (λmax of GLZ) and 234 nm (λmax of MET) as two analytical wavelengths for both drugs in mixture of Water and Methanol (60:40) solvent. 2) Second method based on an K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 58

40 equation of area calculation of curve at two wavelength region (233 to 223nm and 239 to 229 nm). Linearity: Gliclazide: 2 24 μg/ml Metformin: 2 14 μg/ml 5. Gliclazide and Simultaneous It employs formation and 184 Metformin Equation Method solving of simultaneous Hydrochloride equation using two in bulk and wavelengths nm and tablet dosage nm. form Linearity: Gliclazide: 5 25 μg/ml Metformin: μg/ml Table 2.11: Reported methods for estimation of Metformin and Glipizide Sr. Drug/Sample Method Description 1. Metformin and Liquid Column: Zorbax Extend C 18 Glipizide in Chromatography- Mobile phase: ACN: Water: human plasma Tandem Mass Formic acid (70:30: 0.3, v/v/v) Spectrometry Flow Rate: 0.50 ml/min A tandem mass spectrometer equipped with atmospheric pressure chemical ionization source was used as detector and operated in the positive ion mode. 2. Metformin and Liquid The MS/MS detection was Glipizide in Chromatography- performed in multiple Ref K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 59

41 human plasma Tandem Mass reactions monitoring (MRM) Spectrometry mode. 3. Metformin in Its Micellar Liquid Column: Zorbax XDB C cm 187 Multicomponent Chromatography- λ max: 226 nm dosage forms Tandem Mass with Glipizide Spectrometry and Gliclazide 4. Metformin and Liquid The method was applied for 188 Glipizide, Chromatography analysis of Metformin in multi- Gliclazide, component formulations. Glibenclamide Describes the development of or Glimperide in SPE and HPLC methods for the plasma simultaneous determination of Metformin and Glipizide, Gliclazide, Glibenclamide or Glimperide in plasma. 5. Metformin in its ION-pair Liquid HPLC method was developed 189 multicomponent Chromatography for the simultaneous dosage forms estimation of Metformin with Gliclazide and Glipizide present in multicomponent dosage forms. Column: Inertsil C 18 Mobile phase: ACN: Water containing camphor sulphonic acid (adjusted to ph 7 using 0.1 N sodium hydroxide; 75 mm) Flow Rate: 1 ml/min λ max: 225 nm 6. Six sulfonylureas Liquid A method is described for the 190 in serum Chromatography/ separation, identification and K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 60

42 (Carbutamide, Mass Spectrometry quantification of six Tolbutamide, with Atmospheric- sulfonylureas in serum by APCI Glipizide, Pressure Chemical- LC/MS. The sample Glibornuride, Ionization (APCI pretreatment is based on Glibenclamide LC/MS) single-step liquid-liquid and Gliquidone) extraction, using the 4- methylcyclohexyl analogue of Glibenclamide as internal standard. This method allows a screening of these sulfonylureas in serum and subsequently the quantification of the serum level in one run of measurement. 7. Glipizide and UV- Glipizide and Metformin in 191 Metformin in Spectrophotometry combined tablet formulation bulk and its were estimated by using the dosage form multi component mode at 276 nm for Glipizide and 237 nm for Metformin in their solution in methanol. Linearity: Glipizide: 2 20 μg/ml Metformin: 2 20 μg/ml Sr. Table 2.12: Reported methods for estimation of Metformin and Pioglitazone Drug/Sample Method Description Ref. 1. Pioglitazone Derivative Second-derivative responses at 192 Hydrochloride Spectrophotometry nm for PIO and K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 61

43 and Metformin Hydrochloride in tablets and Liquid Chromatography nm for MET in spectra of their solutions in a mixture of methanol and ACN (30:70, v/v) Linearity: Pioglitazone: 8 40 μg/ml (r = ) Metformin: 4 12 μg/ml (r = ) In the LC method Column: ODS-C 18 with 5 μm Mobile phase: ACN: Water: Acetic acid (75: 25: 0.3, v/v/v), adjusted to ph 5.5 with liquor ammonia Flow Rate: 0.5 ml/min Retention time: PIO: 8.5 min MET: 16.0 min Linearity: Pioglitazone: 4 20 μg/ml (r = ) Metformin: 4 20 μg/ml (r = ) λ max: 230 nm 2. Metformin Hydrochloride and Pioglitazone Hydrochloride RP-HPLC Mobile phase: ACN: Water: Acetic acid (60:40:0.3, v/v/v) and the ph was adjusted to 5.5 by adding TEA Flow Rate: 0.5 ml/min Linearity: Pioglitazone: K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 62

44 mg/ml (r = ) Metformin: mg/ml (r = ) 3. Pioglitazone and HPLC Column: Zorbax XDB C 18, 15 cm 194 Metformin in Mobile phase: Buffer: ACN pharmaceutical- (66:34, v/v) of ph 7.1, adjusted dosage form with OPA Buffer: 10 mm disodium hydrogen phosphate and 5mM sodium dodecyl sulphate in double-distilled water Flow Rate: 1 ml/min Column temperature:40 C λ max: 226 nm Internal Standard: Methyl Paraben 4. Pioglitazone Liquid Column: Symmetry Waters C Hydrochloride Chromatography (150 mm 4.6 mm, 5 μm) and Metformin Mobile phase: Potassium Hydrochloride dihydrogen phosphate buffer ph (4.6): ACN (60:40, v/v) Flow Rate: 1 ml/min λ max: 210 nm Retention time: 10 mins Linearity: Pioglitazone: μg/ml Metformin: μg/mL 5. Pioglitazone and Difference Difference spectrum obtained, 196 Metformin in Spectrophotometry by keeping Pioglitazone and bulk drug Metformin separately in 0.1 M and in NaOH in sample cell and 0.1 M K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 63

45 pharmaceutical HCl as blank, showed formulations characteristic peaks (λmax) at 228.1nm (PIO) and 228.2nm (MET) and the characteristics peaks for pharmaceutical formulations were also found. 6. Metformin Ultraviolet The methods employed are 197 Hydrochloride Spectrophotometry derivative spectrophotometery and and Q analysis. The absorption Pioglitazone maxima at 231 nm and 269 nm Hydrochloride in were used for the estimation of a bilayered Metformin and Pioglitazone, tablet dosage respectively. form Linearity: Metformin: μg/ml Pioglitazone: μg/ml 7. Pioglitazone UV- Two Methods: 198 Hydrochloride Spectrophotometry 1) Vierodt s Method which and Metformin involves the formation and Hydrochloride in solving of simultaneous tablets equations at 225 nm and 237 nm, as absorbance maxima of Pioglitazone and Metformin, respectively. 2) Absorption Correction Method which involves direct estimation of Pioglitazone at 267 nm, as at this wavelength Metformin has zero absorbance and shows no interference. For K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 64

46 estimation of Metformin, corrected absorbance was calculated at nm due to the interference of Pioglitazone at this wavelength. Linearity: Metformin: 6 14 μg/ml Pioglitazone: 1 5 μg/ml 8. Metformin Spectrophotometri A method was developed for 199 Hydrochloride, c Multi component simultaneous estimation of Pioglitazone Method Metformin HCl, Pioglitazone Hydrochloride HCl and Glibenclamide in pure and and tablet dosage form by Glibenclamide in using methanol as a solvent. pure and tablet Metformin HCl, Pioglitazone dosage form HCl and Glibenclamide show absorbance maxima at 237 nm, 270 nm and 230 nm respectively. 9. Metformin, Liquid 1) HPLC Method: 200 Pioglitazone and Chromatography Column: Phenomenex RP-18 Glimepiride in and UV Derivative (150 mm x 4.6mm, 5 μm) pharmaceutical Spectrophotometry Mobile phase: ACN: formulations Phosphate buffer (ph 3) (65: 35, v/v) Flow Rate: 0.5 ml/min λ max: 245 nm Run time: 10 min 2) First derivative UV spectrophotometric method K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 65

47 was performed at 260.1, and 251.5nm for MFN, PLZ and GLM respectively. Table 2.13: Reported methods for estimation of Metformin and Sitagliptin Sr. Drug/Sample Method Description 1. Metformin and Laser Diode A simple, rapid and robust Sitagliptin from Thermal high-throughput assay for the mouse and Desorption Tandem simultaneous analysis of human dried Mass Spectrometry Metformin and Sitagliptin from blood spots (LDTD APCI mouse and human dried blood MS/MS) spot samples using LDTD APCI MS/MS was developed for use in a pharmaceutical discovery environment as an alternative to traditional plasma analysis. Analytes were extracted from dried blood spots using a simple punch disc and solvent extract procedure. 2. Metformin and UV Spectroscopy Estimation was carried out by Sitagliptin in multi-component mode of tablet dosage analysis at selected wavelength form of 232 nm and 267 nm for Metformin HCl and Sitagliptin respectively in distilled water. Linearity: 1 40 μg/ml 3. Sitagliptin either Liquid Two Methods: alone or in Chromatography 1) The first method comprised ternary mixture the determination of STG Ref K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 66

48 with Metformin and Sitagliptin degradation product alone in bulk and plasma; and in its pharmaceutical preparation Column: Symmetry Waters C 18 (150 mm 4.6 mm, 5 μm) Mobile phase: Potassium dihydrogen phosphate buffer ph (7.8): ACN (70:30, v/v) Flow Rate: 1 ml/min Fluorescence Detection: λ exi: 267 nm λ emi: 575 nm Linearity: μg/ ml 2) In the second method, the simultaneous determination of Sitagliptin and Metformin in the presence of Sitagliptin alkaline degradation product (SDP) has been developed. Column: Symmetry Waters C 18 (150 mm 4.6 mm, 5 μm) Mobile phase: Potassium dihydrogen phosphate buffer ph (4.6): ACN: Methanol (30:50:20, v/v/v) Flow Rate: 1 ml/min λ max: 220 nm Linearity: Sitagliptin: μg/ml Metformin: μg/ml K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 67

49 4. Metformin HCl and Sitagliptin Phosphate in tablet dosage forms 5. Sitagliptin Phosphate Monohydrate and Metformin Hydrochloride in tablets 6. Sitagliptin in binary mixture with Metformin and ternary mixture RP-HPLC Column: Xterra C 18 (250 mm x 4.6 mm, 5 µm) Mobile Phase: 0.03 M K 2 HPO 4 in water ph-3.2 adjusted with o-phosphoric Acid: ACN in the gradient mode Flow Rate: 1.0 ml/min λ max: 207 nm Retention Time: Metformin: min Sitagliptin: min Linearity: Metformin: µg/ml Sitagliptin: µg/ml UPLC Column: Aquity UPLC BEH C 8 (100 mm x 2.1 mm, 1.7 μm) Mobile Phase: 10 mm Potassium dihydrogen phosphate and 2 mm hexane- 1-sulfonic acid sodium salt (ph adjusted to 5.50 with diluted phosphoric acid) and ACN as organic solvent in a gradient program Flow Rate: 0.2 ml/min λ max: 210 nm Spectroflourometry Four Methods: and 1) Zero order Spectrophotometry spectrophotometric method was used for the determination of STG in the K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 68

50 with Metformin range of μg/ ml. and 2) First derivative Sitagliptin spectrophotometric method alkaline was used for the degradation determination of MET in the product range of 2 12 μg/ml and STG in the range of μg/ml by measuring the peak amplitude at nm and 275 nm, respectively. 3) First derivative of ratio spectra spectrophotometric method used the peak amplitudes at 232 nm and 239 nm for the determination of MET in the range of 2 12 μg/ml. 4) Flourometric method was used for the determination of STG in the range of μg/ml. 7. Sitagliptin and Spectrophotometry Two Methods: 207 Metformin in 1) Direct Spectrophotometric their Method: The first method pharmaceutical was based on measuring the formulation absorbance of Sitagliptin at 268 nm in the range of μg/ml. 2) Isosbestic Method: The second method was the isosbestic point method. The K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya, Gandhinagar 69