Kinetics study of metaxalone degradation under hydrolytic,oxidative and thermal stress conditions using stability-indicating HPLC method

An isocratic stability indicating RP-HPLC–UV method is presented for the determination of metaxalone (MET) in the presence of its degradation products. The method uses Dr. Maisch C18 column (250 mm×4.6 mm, 5 μm) with mobile phase consisting of acetonitrile–potassium dihydrogen orthophosphate buffer with 4 mL of 0.4% triethyl amine (pH 3.0; 10 mM) (58:42, v/v) at a flow rate of 1.0 mL/min. pH of the buffer was adjusted with o-phosphoric acid. UV detection was performed at 225 nm. The method was validated for specificity, linearity, precision, accuracy, limit of detection, limit of quantification and robustness. The calibration plot was linear over the concentration range of 1–100 μg/mL having a correlation coefficient (r²) of 0.999. Limits of detection and quantification were 0.3 and 1 μg/mL, respectively. Intra-day and inter-day precision (% RSD) was 0.65 and 0.79 respectively. The proposed method was used to investigate the degradation kinetics of MET under different stress conditions employed. Degradation of MET followed a pseudo-first-order kinetics, and rate constant (K), time left for 50% potency (t_1/2), and time left for 90% potency (t₉₀) were calculated.     

Development and validation of the stability-indicating LC–UV method for the determination of Cefditoren pivoxil

An isocratic RP-HPLC method was developed for the determination of Cefditoren pivoxil in pharmaceutical formulations using a C-18 column with water–acetonitrile (50:50, v/v) as mobile phase and flow rate 1.2 mL/min (UV detection at 218 nm). Linearity was observed in the concentration range 1.0–250 μg/mL (R²=0.999) with regression equation y=24194x+10749. The forced degradation studies were performed by using HCl, NaOH, and H₂O₂, and thermal and UV radiation. Cefditoren pivoxil is more sensitive towards oxidation and alkaline conditions and resistant towards acidic and photolytic degradations. The method was validated as per ICH guidelines.     

Simultaneous determination of telmisartan and amlodipine in human plasma by LC–MS/MS and its application in a human pharmacokinetic study

A rapid and sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay method has been developed and fully validated for the simultaneous quantification of telmisartan and amlodipine in human plasma. Carbamazepine was used as an internal standard. Analytes and the internal standard were extracted from human plasma by solid-phase extraction technique using Waters Oasis^® HLB 1 cm³ (30 mg) extraction cartridge. The reconstituted samples were chromatographed on a Hypurity advance C₁₈ column (50 mm×4.6 mm, 5 μm) using a mixture of acetonitrile–5 mM ammonium acetate buffer (pH-4.0) (50:50, v/v) as the mobile phase at a flow rate of 0.8 mL/min. The calibration curve obtained was linear (r≥0.99) over the concentration range of 2.01–400.06 ng/mL for telmisartan and 0.05–10.01 ng/mL for amlodipine. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The proposed method was found to be applicable to clinical studies.     

LC-MS determination and pharmacokinetic study of salidroside in rat plasma after oral administration of suspensions of traditional Chinese medicine Erzhi Wan and Fructus Ligustri lucidi

A simple, rapid and sensitive liquid chromatography-mass spectrometry (LC-MS) method was developed for the determination of salidroside in rat plasma and study of its pharmacokinetics after oral administration of suspension of Erzhi Wan and Fructus Ligustri lucidi into Wistar rats. Plasma sample of 200 μL was extracted with acetic ether-isopropanol (2:1) and the extraction was performed on a Kromasil C₁₈ column (150 mm × 4. 6 mm, 5 μm) with the mobile phase of methanol-water (41:59, v/v) within a run time of 6.0 min. The analyte was monitored with positive electrospray ionization (ESI) by selected ion monitoring (SIM) mode. The target ions were m/z 323.05 for salidroside and m/z 411.05 for internal Standard (IS) geniposide. A good linear relationship was obtained over the range of 5.0–500.0 ng/mL and the lower limit of quantification was 5.0 ng/mL. The validated method was successfully applied to the pharmacokinetic study of salidroside in rat plasma after oral administration of suspension of Erzhi Wan and Fructus Ligustri lucidi.     

Development of a sensitive and rapid method for quantitation of (S)-(−)- and (R)-(+)-metoprolol in human plasma by chiral LC–ESI–MS/MS

A selective, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC–ESI–MS/MS) method has been developed for separation and quantification of metoprolol enantiomers on a chiral Lux Amylose-2 (250 mm×4.6 mm, 5 μm) column. Solid phase extraction of (S)-(−)- and (R)-(+)-metoprolol and rac-metoprolol-d6 as an internal standard (IS) was achieved on Lichrosep DVB HL cartridges employing 200 μL human plasma. Both the analytes were chromatographically separated with a resolution factor of 2.24 using 15 mM ammonium acetate in water, pH 5.0 and 0.1% (v/v) diethyl amine in acetonitrile (50:50, v/v) as the mobile phase within 7.0 min. The precursor→product ion transitions for the enantiomers and IS were monitored in the multiple reaction monitoring and positive ionization mode. The method was validated over the concentration range of 0.500–500 ng/mL for both the enantiomers. Matrix effect was assessed by post-column analyte infusion experiment and the mean extraction recovery was greater than 94.0% for both the enantiomers at all quality control levels. The stability of analytes was evaluated in plasma and whole blood under different storage conditions. The method was successfully applied to a clinical study in 14 healthy volunteers after oral administration of 200 mg metoprolol tablet under fasting conditions. The assay reproducibility is shown by reanalysis of 68 incurred samples. The suitability of the developed method was assessed in comparison with different chromatographic methods developed for stereoselective analysis of metoprolol in biological matrices.     

Quantification of desloratadine in human plasma by LC-ESI-MS/MS and application to a pharmacokinetic study

A simple, sensitive, and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of desloratadine (DL) in human plasma using desloratadine-d5 (DLD5) as an internal standard (IS). Chromatographic separation was performed using an Xbridge C18 column (50 mm×4.6 mm, 5 μm) with an isocratic mobile phase composed of 10 mM ammonium formate: methanol (20:80, v/v), at a flow rate of 0.7 mL/min. DL and DLD5 were detected with proton adducts at m/z 311.2→259.2 and 316.2→264.3 in multiple reaction monitoring (MRM) positive modes, respectively. Liquid–liquid extraction (LLE) method was used to extract the drug and the IS. The method was validated over a linear concentration range of 5.0–5000.0 pg/mL with a correlation coefficient of (r²)≥0.9994. This method demonstrated intra- and inter-day precision within 0.7–2.0% and 0.7–2.7%, and an accuracy within 101.4–102.4%, and 99.5–104.8%. DL was found to be stable throughout the freeze–thaw cycles, bench-top, and postoperative stability studies. This method was successfully applied in the analysis of plasma samples following oral administration of DL (5 mg) in 35 healthy Indian male human volunteers under fasting conditions.     

Validation and application by HPLC for simultaneous determination of vitexin-2″-O-glucoside,vitexin-2″-O-rhamnoside,rutin, vitexin,and hyperoside

A simple, precise, and rapid high-performance liquid chromatographic method was developed and validated for the simultaneous determination of vitexin-2″-O-glucoside, vitexin-2″-O-rhamnoside, rutin, vitexin, and hyperoside. The HPLC separation was performed using a Shim-pack VP-ODS C₁₈ column (250 mm×4.6 mm i.d., 5 μm) with the isocratic mobile phase consisting of tetrahydrofuran/ acetonitrile/0.05% phosphoric acid solution (20:3:77, v/v/v), and the flow rate was set at 1.0 mL/min. UV detection was carried out at a wavelength of 360 nm and the whole analysis took 25 min. The method was linear in the range of 4.12–206.00 μg/mL for vitexin-2″-O-glucoside, 4.05–202.50 μg/mL for vitexin-2″-O-rhamnoside, 1.64–82.00 μg/mL for rutin, 1.74–87.00 μg/mL for vitexin, and 1.41–70.60 μg/mL for hyperoside with the correlation coefficient for each analyte more than 0.998. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.6 and 2 ng for vitexin-2″-O-glucoside, 0.6 and 2 ng for vitexin-2″-O-rhamnoside, 0.3 and 1 ng for rutin, 1 and 3 ng for vitexin, and 0.5 and 2 ng for hyperoside, respectively. Intra- and inter-day precision and accuracy (RSD) were less than 3%. The developed HPLC method was successfully applied to the analysis of five flavonoids in hawthorn leaves, hawthorn fruits, and the preparations containing hawthorn leaves or fruits.     

Analysis of in vivo absorption of didanosine tablets in male adult dogs by HPLC

Didanosine is an effective antiviral drug in untreated and antiretroviral therapy-experienced patients with Human Immunodeficiency Virus (HIV). An automated system using on-line solid extraction and High Performance Liquid Chromatography (HPLC) with ultraviolet (UV) detection was developed and validated for pharmacokinetic analysis of didanosine in dog plasma. Modifications were introduced on a previous methodology for simultaneous analysis of antiretroviral drugs in human plasma. Extraction was carried out on C18 cartridges, with high extraction yield as stationary phase, whereas mobile phase consisted of a mixture of 0.02 M potassium phosphate buffer, acetonitrile (KH₂PO₄: acetonitrile: 96:4, v/v) and 0.5% (w/v) of heptane sulphonic acid. The pH was adjusted to 6.5 with triethylamine. All samples and standard solutions were chromatographed at 28 °C. For an isocratic run, the flux was 1.0 mL/min, detection was at 250 nm and injected volume was 20 μL. The method was selective and linear for concentrations between 50 and 5000 ng/mL. Drug stability data ranged from 96% to 98%, and limit of quantification was 25 ng/mL. Extraction yield was up to 95%. Drug stability in dog plasma was kept frozen at ?20 °C for one month after three freeze–thaw cycles, and for 24 h after processing in the auto sampler. Assay was successfully applied to measure didanosine concentrations in plasma dogs.     

Determination of metabolite of nicergoline in human plasma by high-performance liquid chromatography and its application in pharmacokinetic studies

A fast, simple and sensitive high performance liquid chromatographic (HPLC) method has been developed for determination of 10α-methoxy-6-methyl ergoline-8β-methanol (MDL, a main metabolite of nicergoline) in human plasma. One-step liquid–liquid extraction (LLE) with diethyl ether was employed as the sample preparation method. Tizanidine hydrochloride was selected as the internal standard (IS). Analysis was carried out on a Diamonsil ODS column (150 mm×4.6 mm, 5 μm) using acetonitrile–ammonium acetate (0.1 mol/L) (15/85, v/v) as mobile phase at detection wavelength of 224 nm. The calibration curves were linear over the range of 2.288–73.2 ng/mL with a lower limit of quantitation (LLOQ) of 2.288 ng/mL. The intra- and inter-day precision values were below 13% and the recoveries were from 74.47% to 83.20% at three quality control levels. The method herein described was successfully applied in a randomized crossover bioequivalence study of two different nicergoline preparations after administration of 30 mg in 20 healthy volunteers.     

Development and validation of a normal-phase HPTLC method for the simultaneous analysis of Lamivudine and Zidovudine in fixed-dose combination tablets

Simultaneous quantification of Lamivudine and Zidovudine in tablets by HPTLC method was developed and validated. The chromatograms were developed using a mobile phase of toluene:ethyl acetate:methanol (4:4:2, v/v/v) on pre-coated plate of silica gel GF aluminum TLC plate and quantified by densitometric absorbance mode at 276 nm. The R_f values were 0.41±0.03 and 0.60±0.04 for Lamivudine and Zidovudine, respectively. The linearity of the method was found to be within the concentration range of 50?250 ng/spot for Lamivudine and for Zidovudine, it was 100?500 ng/spot. The lower limits of detection and quantification were 2.23 ng/spot and 7.90 ng/spot for Lamivudine and 2.90 ng/spot and 8.85 ng/spot for Zidovudine. The method was also validated for precision, specificity and recovery. This developed method was used to analyze fixed-dose tablets (Duovir, Cipla Ltd) samples of Lamivudine and Zidovudine.     

New chiral reverse phase HPLC method for enantioselective analysis of ketorolac using chiral AGP column

A simple, specific, precise, sensitive and rapid reverse phase-HPLC method was developed for determination of ketorolac enantiomers, a potent nonnarcotic analgesic in pharmaceutical formulations. The method was developed on a chiral AGP column. Mobile phase was 0.1 M sodium phosphate buffer (pH 4.5): Isopropanol (98:2, v/v), at a flow rate of 1 mL/min with run time of 15 min. Ultraviolet detection was made at 322 nm. The linearity range was 0.02–10 μg/mL for each of the enantiomers. The mobile phase composition was systematically studied to find the optimum chromatographic conditions. Validation of the method under the conditions selected showed that it was selective and precise and that the detector response was linear function of ketorolac.     

Determination of genotoxic alkyl methane sulfonates and alkyl paratoluene sulfonates in lamivudine using hyphenated techniques

Two highly sensitive methods for the determination of genotoxic alkyl methane sulfonates (AMSs) and alkyl paratoluene sulfonates (APTSs) in lamivudine using hyphenated techniques have been presented. AMSs were determined by GC–MS method using GSBP-INOWAX (30 m×0.25 mm×0.25 μm) column. Temperature program was set by maintaining at 100 °C initially for 3 min, then rised to 220 °C at the rate of 15 °C/min and maintained at 220 °C for 16 min. N,N-dimethyl formamide was used as diluent. APTSs were determined by LC-MS using Zorbax, Rx C8, 250 mm×4.6 mm, 5 μm column as stationary phase. 0.01 M ammonium acetate is used as buffer. The mixture of buffer and methanol in 75:25 (v/v) ratio was used as mobile phase A and mixture of buffer and methanol in 5:95 (v/v) ratio was used as mobile phase B. The gradient program (T/%B) was set as 0/28, 16/50, 17/100, 23/100, 27/28 and 40/28. Both the methods were validated as per International Conference on Harmonization guidelines. Limit of quantitation was found 1.5 μg/mL for AMSs and was in the range of 1.0–1.5 μg/mL for APTSs.     

Simultaneous determination of pioglitazone and candesartan in human plasma by LC-MS/MS and its application to a human pharmacokinetic study

A simple and rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and fully validated for simultaneous quantification of pioglitazone and candesartan in human plasma. Irbesartan was used as an internal standard. The analytes were extracted from human plasma samples by solid-phase extraction technique using a Strata-X 33 μm polymeric sorbent. The reconstituted samples were chromatographed on a C₁₈ column by using a 80:20 (v/v) mixture of acetonitrile and 0.1% formic acid as the mobile phase at a flow rate of 0.8 mL/min. The calibration curves obtained were linear (r≥0.99) over the concentration range of 15–3000 ng/mL for pioglitazone and 5–608 ng/mL for candesartan. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. A run time of 2.7 min for each sample made it possible to analyze more than 300 plasma samples per day. The proposed method was found to be applicable to clinical studies.     

Development and validation of analytical method for the estimation of nateglinide in rabbit plasma

Nateglinide has been widely used in the treatment of type-2 diabetics as an insulin secretogoga. A reliable, rapid, simple and sensitive reversed-phase high performance liquid chromatography (RP-HPLC) method was developed and validated for determination of nateglinide in rabbit plasma. The method was developed on Hypersil BDSC-18 column (250 mm×4.6 mm, 5 mm) using a mobile phase of 10 mM phosphate buffer (pH 2.5) and acetonitrile (35:65, v/v). The elute was monitored with the UV–vis detector at 210 nm with a flow rate of 1 mL/min. Calibration curve was linear over the concentration range of 25–2000 ng/mL. The retention times of nateglinide and internal standard (gliclazide) were 9.608 min and 11.821 min respectively. The developed RP-HPLC method can be successfully applied to the quantitative pharmacokinetic parameters determination of nateglinide in rabbit model.     

High performance liquid chromatography mass spectrometric method for the simultaneous quantification of pravastatin and aspirin in human plasma: Pharmacokinetic application

A rapid and sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay method has been developed and fully validated for the simultaneous quantification of pravastatin and aspirin in human plasma. Furosemide was used as an internal standard. Analytes and the internal standard were extracted from human plasma by liquid–liquid extraction technique using methyl tertiary butyl ether. The reconstituted samples were chromatographed on a Zorbax SB-C₁₈ column by using a mixture of 5 mM ammonium acetate buffer and acetonitrile (20:80, v/v) as the mobile phase at a flow rate of 0.8 mL/min. The calibration curve obtained was linear (r≥0.99) over the concentration range of 0.50–600.29 ng/mL for pravastatin and 20.07–2012.00 ng/mL for aspirin. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 2.0 min for each sample made it possible to analyze more than 400 human plasma samples per day. The proposed method was found to be applicable to clinical studies.     

Simultaneous determination of nortriptyline hydrochloride and fluphenazine hydrochloride in microgram quantities from low dosage forms by liquid chromatography–UV detection

A novel method for the simultaneous high-performance liquid chromatographic determination of nortriptyline hydrochloride and fluphenazine hydrochloride was developed and validated. Fluvastatin sodium was used as internal standard. The determination was performed on a Hypersil Gold C₈ column (250 mm × 4.6 mm i.d., 5 μm particle size) at 25 °C; the mobile phase, consisting of a mixture of formic acid (0.1 M, pH 2.16)-methanol (33:67, v/v), was delivered at a flow rate of 1.1 mL/min and detector wavelength at 251 nm. The retention time of nortriptyline, fluphenazine and fluvastatin was found to be 5.11, 8.05 and 11.38 min, respectively. Linearity ranges were 5.0–1350.0 and 10.0–1350.0 μg/mL with limit of detection values of 0.72 and 0.31 μg/mL, for nortriptyline and fluphenazine, respectively. Results of assay and recovery studies were statistically evaluated for its accuracy and precision. Correlation coefficients (r²) of the regression equations were greater than 0.999 in all cases. According to the validation results, the proposed method was found to be specific, accurate, precise and could be applied to the simultaneous quantitative analysis of nortriptyline and fluphenazine.     

Development and validation of analytical method for the estimation of lamivudine in rabbit plasma

Lamivudine has been widely used in the treatment of HIV disease. A reliable, sensitive reversed phase high performance liquid chromatography (RP-HPLC) method was developed and validated for lamivudine in rabbit plasma. The method was developed on Hypersil BDS C-18 column (250 mm×4.6 mm, 5 μm) using a mobile phase of 0.25% Triethylamine buffer (pH 3.0): acetonitrile (70:30, v/v). The efficient was monitored by UV detector at 256 nm. The total run time was 15 min with a flow rate of 1.0 mL/min. Calibration curve was linear over the concentration range of 25–2000 ng/mL. The retention times of lamivudine and internal standard (Nelfinavir) were 8.78 min and 10.86 min, respectively. The developed RP-HPLC method can be successfully applied for the quantitative pharmacokinetic parameters determination of lamivudine in rabbit model.     

Cleaning level acceptance criteria and HPLC-DAD method validation for the determination of Nabumetone residues on manufacturing equipment using swab sampling

Prevention of cross contamination with active pharmaceutical ingredients is crucial and requires special attention in pharmaceutical industries. Current method validation describes the determination of Nabumetone (NAB) residue on a stainless steel surface using swab sampling with a sensitive HPLC-DAD analysis. The acceptance limit was decided as 2 μg swab per 100 cm². Cotton swabs impregnated with extraction solution were used to determine residual drug content. Recoveries were 90.88%, 91.42%, and 92. 21% with RSD ranging from 2.2% to 3.88% at three concentration levels. Residual concentration was found to be linear in the range of 0.1–4.56 μg/mL, when estimated using a Phenomenex Luna C₁₈ (25 cm×5 μm×4.6 mm i.d.) column at 1.0 mL/min flow rate and 230 nm. The mobile phase consisted of a mixture of methanol:acetonitrile:water (55:30:15, v/v/v). The LOD and LOQ for NAB were found to be 0.05 and 0.16 μg/mL, respectively. The validated method was found to be simple, selective and sensitive for demonstration of cleaning validation of NAB residues on a stainless steel surface.     

Improved simultaneous quantitation of candesartan and hydrochlorthiazide in human plasma by UPLC–MS/MS and its application in bioequivalence studies

A validated ultra-performance liquid chromatography mass spectrometric method (UPLC–MS/MS) was used for the simultaneous quantitation of candesartan (CN) and hydrochlorothiazide (HCT) in human plasma. The analysis was performed on UPLC–MS/MS system using turbo ion spray interface. Negative ions were measured in multiple reaction monitoring (MRM) mode. The analytes were extracted using a liquid–liquid extraction (LLE) method by using 0.1 mL of plasma volume. The lower limit of quantitation for CN and HCT was 1.00 ng/mL whereas the upper limit of quantitation was 499.15 ng/mL and 601.61 ng/mL for CN and HCT respectively. CN d₄ and HCT-¹³Cd₂ were used as the internal standards for CN and HCT respectively. The chromatography was achieved within 2.0 min run time using a C18 Phenomenex, Gemini NX (100 mm×4.6 mm, 5 µm) column with organic mixture:buffer solution (80:20, v/v) at a flow rate of 0.800 mL/min. The method has been successfully applied to establish the bioequivalence of candesartan cilexetil (CNC) and HCT immediate release tablets with reference product in human subjects.     

The clinical value of enzyme-multiplied immunoassay technique monitoring the plasma concentrations of cyclosporine A after renal transplantation

The feasibility and the clinical value of the enzyme-multiplied immunoassay technique (EMIT) monitoring of blood concentrations of cyclosporine A (CsA) in patients treated with CsA were investigated after kidney transplantation. The validation method was performed to the EMIT determination of CsA blood concentration, the CsA whole blood ‘trough concentrations (C₀) of patients in different time periods after renal transplantation were monitored, and combined with the clinical complications, the statistical results were analyzed and compared. EMIT was precise, accurate and stable, also with a high quality control. The mean postoperative blood concentration of CsA was as follows: <1 month, (281.4 ± 57.9)ng/mL; 2 – 3 months, (264.5 ± 41. 2)ng/mL; 4 – 5 months, (236.4 ± 38. 9)ng/mL; 6 – 12 months, (206.5 ± 32.6)ng/mL; >12 months, (185.6 ± 28.1)ng/mL. The toxic reaction rate of CsA blood concentration within the recommended therapeutic concentration was 14. 1%, significantly lower than that of the none-recommended dose group (37.2%) (P < 0.05); the transplantation rejection rate was 4.4%, significantly lower than that of the none-recommended dose group (22.5%) (P < 0.05). Using EMIT to monitor the blood concentration of CsA as the routine laboratory method is feasible, and is able to reduce the CsA toxicity and rejection significantly, leading to achieving the desired therapeutic effect.