High performance liquid chromatographic separation of thirteen drugs collected in Chinese Pharmacopoeia 2010(Ch.P2010) on cellulose ramification chiral stationary phase

The enantiomers separation of thirteen drugs collected in Ch.P2010 was performed on chiral stationary phase of cellulose ramification (chiralpak OD and chiralpak OJ) by high performance liquid chromatographic (HPLC) methods, which included ibuprofen (C1), ketoprofen (C2), nitrendipine (C3), nimodipine (C4), felodipine (C5), omeprazole (C6), praziquantel (C7), propranolol hydrochloride (C8), atenolol (C9), sulpiride (C10), clenbuterol hydrochloride (C11), verapamil hydrochloride (C12), and chlorphenamine maleate (C13). The mobile phase consisted of isopropanol and n-hexane. The detection wavelength was set at 254 nm and the flow rate was 0.7 mL/min. The enantiomers separation of these thirteen racemates on chiralpak OD column and chiralpak OJ column was studied, while the effects of proportion of organic additives, alcohol displacer and temperature on the separation were studied. And the mechanism of some of racemates was discussed. The results indicated that thirteen chiral drugs could be separated on chiral stationary phase of cellulose ramification in normal phase chromatographic system. The chromatographic retention and resolution of enantiomers could be adjusted by factors including column temperature and the concentration of alcohol displacer and organic alkaline modifier in mobile phase. It was shown that the resolution was improved with reducing concentration of alcohol displacer. When concentration of organic alkaline modifier was 0.2% (v/v), the resolution and the peak shape were fairly good. Most racemates mentioned above had better resolution at column temperature of 25 °C. When racemates were separated, the temperature should be kept so as to obtain stable separation results.     

Development and validation of RP-HPLC method for estimation of eplerenone in spiked human plasma

A rapid and simple high performance liquid chromatography (HPLC) method with a UV detection (241 nm) was developed and validated for estimation of eplerenone from spiked human plasma. The analyte and the internal standard (valdecoxib) were extracted with a mixture of dichloromethane and diethyl ether. The chromatographic separation was performed on a HiQSil C-18HS column (250 mm×4.6 mm, 5 μm) with a mobile phase consisting of acetonitrile:water (50:50, v/v) at flow rate of 1 mL/min. The calibration curve was linear in the range 100–3200 ng/mL and the heteroscedasticity was minimized by using weighted least squares regression with weighting factor 1/X.     

Development and validation of RP-HPLC and RP-UPLC methods for quantification of parathyroid hormones (1-34) in medicinal product formulated with meta-cresol

Rapid and sensitive reversed phase high performance liquid chromatography (RP-HPLC) and ultra performance liquid chromatography (RP-UPLC) method with UV detection has been developed and validated for quantification of parathyroid hormone (PTH) in presence of meta-cresol as a stabilizer in a pharmaceutical formulation. Chromatography was performed with mobile phase containing 0.1% Trifluoroacetic acid (TFA) in MilliQ water and 0.1% TFA in acetonitrile with gradient program and flow rate at 0.3 mL/min for HPLC and 0.4 mL/min for UPLC. Quantification was accomplished with internal reference standard (qualified against innovator product and National Institute for Biological Standards and Control (NIBSC) standard). The methods were validated for linearity (correlation coefficient=0.99), range, accuracy, precision and robustness. Robustness was confirmed by considering three factors; mobile phase composition, column temperature and flow rate/age of mobile phase.Intermediate precision was confirmed on different equipments, different columns and on different days. The relative standard deviation (RSD) (<2% for RP-HPLC and <1% for UPLC, n=30) indicated a good precision. Retention time was found about 17 min and 2 min by HPLC and UPLC methods, respectively. Both methods are simple, highly sensitive, precise and accurate and have the potential of being useful for routine quality control.     

Development of a validated HPLC method for the separation and analysis of a Bromazepam,Medazepam and Midazolam mixture

The purpose of this work was to develop a rapid, sensitive and validated HPLC method for the separation and analysis of a Bromazepam, Medazepam and Midazolam mixture. The three benzodiazepine compounds were separated on a reversed-phase C18 column at 50 °C using a mobile phase containing 25% acetonitrile, 45% methanol and 30% ammonium acetate (0.05 M). The pH was adjusted to pH=9 by the addition of ammonia solution (35%, w/w). The samples were detected using a UV detector at 240 nm. The validation study of the method included the effect of temperature, flow rate, ratio of the components of the mobile phase and the pH of the mobile phase on the efficiency of separation. The linear range of Bromazepam and Midazolam was between 0.12 and 0.18 mg/mL, while that of Medazepam was between 0.08 and 0.12 mg/mL. The relative standard deviation for precision was less than 2%. The linearity, selectivity, accuracy and robustness of the developed method showed acceptable values. The method was applied to the analysis of the samples of raw material of the three compounds under study, and the percentage of recoveries was 99.89%±1.06. It was also applied to the analysis of samples of pharmaceutical preparations of those compounds and spiked serum samples. Recoveries from serum samples ranged between 91.5% and 99.0%. The developed method is suitable for quality control of Bromazepam, Medazepam and Midazolam in their mixtures and in pharmaceutical preparations (tablets, capsules, ampoules). It can also be used to determine their concentrations in serum.     

Development and validation of RP-HPLC and RP-UPLC methods for quantification of erythropoietin formulated with human serum albumin

Rapid and sensitive reverse phase high performance liquid chromatography (RP–HPLC) and ultra performance liquid chromatography (UPLC) methods with UV detection for quantification of erythropoietin (EPO) in presence of human serum albumin (HSA) as a stabilizer in a pharmaceutical formulation of EPO have been developed and validated. Chromatography was performed with mobile phase containing 0.1% Trifluoroacetic acid (TFA) in MilliQ water and 0.1% TFA in acetonitrile with gradient program and a flow rate of 1.5 mL/min for HPLC and 0.35 mL/min for UPLC. Quantification was accomplished with internal reference standard (qualified using EP reference standard). The methods were validated for linearity (correlation coefficient=0.99), accuracy, precision and robustness. Robustness was confirmed by considering three factors; percentages of TFA in mobile phase, age of test sample and mobile phase and column temperature. Intermediate precision was confirmed by different analysts, different equipments and on different days. The relative standard deviation (RSD) value (<2%, n=30) indicated good precision of the developed method. The proposed RP-HPLC method had retention time less than 20 min while the developed UPLC method had retention time less than 4 min. Both the RP-HPLC and UPLC methods were simple, highly sensitive, precise and accurate, suggesting that the developed methods are useful for routine quality control.     

LC–UV/MS quality analytics of paediatric artemether formulations

A highly selective and stability-indicating HPLC-method, combined with appropriate sample preparation steps, is developed for β-artemether assay and profiling of related impurities, including possible degradants, in a complex powder for oral suspension. Following HPLC conditions allowed the required selectivity: a Prevail organic acid (OA) column (250 mm×4.6 mm, 5 μm), flow rate set at 1.5 mL/min combined with a linear gradient (where A=25 mM phosphate buffer (pH 2.5), and B=acetonitrile) from 30% to 75% B in a runtime of 60 min. Quantitative UV-detection was performed at 210 nm. Acetonitrile was applied as extraction solvent for sample preparation. Using acetonitrile–water mixtures as extraction solvent, a compartmental behaviour by a non-solving excipient-bound fraction and an artemether-solubilising free fraction of solvent was demonstrated, making a mobile phase based extraction not a good choice. Method validation showed that the developed HPLC-method is considered to be suitable for its intended regulatory stability-quality characterisation of β-artemether paediatric formulations. Furthermore, LC–MS on references as well as on stability samples was performed allowing identity confirmation of the β-artemether related impurities. MS-fragmentation scheme of β-artemether and its related substances is proposed, explaining the m/z values of the in-source fragments obtained.     

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.     

Precolumn derivatization LC–MS/MS method for the determination and pharmacokinetic study of glucosamine in human plasma and urine

A selective precolumn derivatization liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the determination of glucosamine in human plasma and urine has been developed and validated. Glucosamine was derivatized by o-phthalaldehyde/3-mercaptopropionic acid. Chromatographic separation was performed on a Phenomenex ODS column (150 mm×4.6 mm, 5 μm) using linear gradient elution by a mobile phase consisting of methanol (A), and an aqueous solution containing 0.2% ammonium acetate and 0.1% formic acid (B) at a flow rate of 1 mL/min. Tolterodine tartrate was used as the internal standard (IS). With protein precipitation by acetonitrile and then the simple one-step derivatization, a sensitive bio-assay was achieved with the lower limit of quantitation (LLOQ) as low as 12 ng/mL for plasma. The standard addition calibration curves suitable for clinical sample analysis showed good linearity over the range of 0.012–8.27 μg/mL in plasma and 1.80–84.1 μg/mL in urine. The fully validated method has been successfully applied to a pharmacokinetic study of compound glucosamine sulfate dispersible tablets in health Chinese volunteers receiving single oral doses at 500, 1000 and 1500 mg of glucosamine sulfate, as well as multiple oral doses of 500 mg t.i.d. for 7 consecutive days.     

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.     

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.     

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.     

Preparative isolation and purification of 12,13-dihydroxyeuparin from Radix Eupatorii Chinensis by high-speed counter-current chromatography

An ef?cient method for the isolation and puri?cation of 12,13-dihydroxyeuparin from Radix Eupatorii Chinensis by high speed counter-current chromatography (HSCCC) was established in this paper. The ether extracts of Radix Eupatorii Chinensis were puri?ed by HSCCC with a solvent system of hexyl hydride–ethyl acetate–methanol–water (1:2:1:2, v/v/v/v). The upper phase was used as the stationary phase and the lower phase as the mobile phase. About 8.4 mg of 12,13-dihydroxyeuparin was obtained from 200 mg of ether extracts from Radix Eupatorii Chinensis in one-step HSCCC separation, with the purity of 96.71%, as determined by HPLC. After methanol–water recrystallization, the purity of 12,13-dihydroxyeuparin reached 99.83%. Such a simple and effective method was fairly useful to prepare pure compound as reference substances for related study on Radix Eupatorii Chinensis.     

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.     

Fingerprint analysis of placenta polypeptide injection by high performance liquid chromatography

Objective: To develop the representative fingerprint for the quality control of placenta polypeptide injection.MethodsThe chromatographic separation was performed using a Phenomenex Gemini C18 column (250 mm×4.6 mm, 5 μm) maintained at 30 °C. 0.1% aqueous trifluoroacetic acid (Solvent A) and acetonitrile contained 0.1% TFA (Solvent B) were used as mobile phase with a gradient elution. Detection wavelength was 280 nm with the sample injection volume of 50 μL; the flow rate was 1.0 mL/min. The fingerprints of different samples were investigated by similarity analysis.ResultsNine peaks were identified as the characteristic common peaks. The similarities of the fingerprints of the 10 batches of samples were above 0.992.ConclusionThis method showed high precision and good repeatability, and provided the basis for the improvement of the quality control of placenta polypeptide injection.     

Determination of mycophenolic acid in human plasma by ultra performance liquid chromatography tandem mass spectrometry

A simple, sensitive and high throughput ultra performance liquid chromatography tandem mass spectrometry method has been developed for the determination of mycophenolic acid in human plasma. The method involved simple protein precipitation of MPA along with its deuterated analog as an internal standard (IS) from 50 µL of human plasma. The chromatographic analysis was done on Acquity UPLC C18 (100 mm×2.1 mm, 1.7 µm) column under isocratic conditions using acetonitrile and 10 mM ammonium formate, pH 3.00 (75:25, v/v) as the mobile phase. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for quantitation. In-source conversion of mycophenolic glucuronide metabolite to the parent drug was selectively controlled by suitable optimization of cone voltage, cone gas flow and desolvation temperature. The method was validated over a wide concentration range of 15–15000 ng/mL. The mean extraction recovery for the analyte and IS was >95%. Matrix effect expressed as matrix factors ranged from 0.97 to 1.02. The method was successfully applied to support a bioequivalence study of 500 mg mycophenolate mofetil tablet in 72 healthy subjects.     

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.     

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.     

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.     

Validated gradient stability indicating HPLC method for determining Diltiazem Hydrochloride and related substances in bulk drug and novel tablet formulation

A stability-indicating liquid chromatographic method has been developed and validated for the determination of Diltiazem Hydrochloride (DTZ) together with its six related substances (Diltiazem sulphoxide, Imp-A, Imp-B, Imp-D, Imp-E, and Imp-F) in a laboratory mixture as well as in a novel tablet formulation developed in-house. Efficient chromatographic separation was achieved on a Hypersil BDS C₁₈ (150 mm×4.6 mm, 5.0 μm) with mobile phase containing 0.2% Triethylamine (TEA) in gradient combination with acetonitrile (ACN) at a flow rate of 1.0 mL/min and the eluent was monitored at 240 nm. In the developed method, the resolution of DTZ from any pair of impurities was found to be greater than 2.0. The test solution and related substances were found to be stable in the diluent for 24 h. The developed method resolved the drug from its known impurities, stated above, and also from additional impurities generated when the formulation was subjected to forced degradation; the mass balance was found close to 99.9%.Regression analyses indicate correlation coefficient value greater than 0.997 for DTZ and its six known impurities. The LOD for DTZ and the known impurities was at a level below 0.02%. The method has shown good, consistent recoveries for DTZ (99.8–101.2%) and also for its six known impurities (97.2–101.3%). The method was found to be accurate, precise, linear, specific, sensitive, rugged, robust, and stability-indicating.     

Selective separation,detection of zotepine and mass spectral characterization of degradants by LC–MS/MS/QTOF

A simple, precise, accurate stability-indicating gradient reversed-phase high-performance liquid chromatographic (RP–HPLC) method was developed for the quantitative determination of zotepine (ZTP) in bulk and pharmaceutical dosage forms in the presence of its degradation products (DPs). The method was developed using Phenomenex C₁₈ column (250 mm×4.6 mm i.d., 5 µm) with a mobile phase containing a gradient mixture of solvents, A (0.05% trifluoroacetic acid (TFA), pH=3.0) and B (acetonitrile). The eluted compounds were monitored at 254 nm; the run time was within 20.0 min, in which ZTP and its DPs were well separated, with a resolution of >1.5. The stress testing of ZTP was carried out under acidic, alkaline, neutral hydrolysis, oxidative, photolytic and thermal stress conditions. ZTP was found to degrade significantly in acidic, photolytic, thermal and oxidative stress conditions and remain stable in basic and neutral conditions. The developed method was validated with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness as per ICH guidelines. This method was also suitable for the assay determination of ZTP in pharmaceutical dosage forms. The DPs were characterized by LC–MS/MS and their fragmentation pathways were proposed.