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.     

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 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.     

Simultaneous determination of oleanolic acid and ursolic acid by RP-HPLC in the leaves of Eriobotrya japonica Lindl.

Oleanolic acid (OA) and ursolic acid (UA) are isomeric triterpenic acids and only one methyl's position is different between them. OA and UA always exist in the same plant, so it is difficult to separate them when determining contents by RP-HPLC. In this study, a very simple mobile phase for HPLC was developed to simultaneously determine UA and OA, and the factors affecting separation were also discussed. The mobile phase is methanol: water (95:5) with flow rate 0.4 mL/min. The retention time for OA and UA was 20.58 and 21.57 min, respectively, the resolution was 1.61. The average contents of OA and UA of three Loquat leaves sets were 1.4 mg/g and 5.6 mg/g, respectively. Regarding the HPLC, we found that changing mobile phase, adjusting the pH value or adding ion-pairing agent could not affect the separation between UA and OA greatly. While adjustment of the flow rate and column temperature could improve the resolution greatly.     

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.     

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.     

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.     

Bio-analytical method development and validation of Rasagiline by high performance liquid chromatography tandem mass spectrometry detection and its application to pharmacokinetic study

The most suitable bio-analytical method based on liquid–liquid extraction has been developed and validated for quantification of Rasagiline in human plasma. Rasagiline-¹³C₃ mesylate was used as an internal standard for Rasagiline. Zorbax Eclipse Plus C18 (2.1 mm×50 mm, 3.5 μm) column provided chromatographic separation of analyte followed by detection with mass spectrometry. The method involved simple isocratic chromatographic condition and mass spectrometric detection in the positive ionization mode using an API-4000 system. The total run time was 3.0 min. The proposed method has been validated with the linear range of 5–12000 pg/mL for Rasagiline. The intra-run and inter-run precision values were within 1.3%–2.9% and 1.6%–2.2% respectively for Rasagiline. The overall recovery for Rasagiline and Rasagiline-¹³C₃ mesylate analog was 96.9% and 96.7% respectively. This validated method was successfully applied to the bioequivalence and pharmacokinetic study of human volunteers under fasting condition.     

LC–UV and LC–MS evaluation of stress degradation behavior of desvenlafaxine

The objective of current study was to develop a validated specific stability indicating reversed-phase liquid chromatographic method for the quantitative determination of desvenlafaxine in bulk sample and pharmaceutical dosage form in the presence of degradation products. Forced degradation studies were performed on bulk sample of desvenlafaxine as per ICH prescribed stress conditions using acid, base, oxidative and photolytic degradation to show the stability indicating power of the method. Significant degradation was observed under acidic stress condition and the degradation product formed was identified by LC–MS and a degradation pathway for drug has been proposed. Successful separation of drug from degradation products formed under stress conditions was achieved on a SymmetryShield column C₁₈ (5 μm, 250 mm×4.6 mm, i.d.) using the mobile phase consisting of a mixture of 0.2% (v/v) triethylamine in ammonium acetate (0.05 M; pH 6.5) and methanol using isocratic gradient.     

Determination and pharmacokinetic study of catechin in rat plasma by HPLC

A high performance liquid chromatographic method was developed and validated for the quantitative determination of catechin in rat plasma and its pharmacokinetic study after intragastric administration of Catechu and Xiongdanjiangre Wan into SD rats. Plasma samples were prepared by protein precipitation using methanol–5% aqueous zinc sulfate (70:30, v/v) as precipitant. Chromatographic separation was achieved on Hypersil C₁₈ column (250 mm×4.6 mm, 10 μm) with acetonitrile–water–triethylamine (6:94:0.3, v/v/v, pH 4.0±0.1, adjusted with phosphoric acid) as mobile phase, followed by a UV detection at 207 nm. Good linearity was obtained over the range of 0.143–7.15 mg/L of catechin, with correlation coefficient of 0.9992. The method was simple, sensitive, accurate and reproducible and has been successfully applied to the pharmacokinetic study of catechin in rat plasma.     

HPLC手性流动相添加剂法分离测定盐酸度洛西汀左旋异构体

建立检查盐酸度洛西汀中左旋异构体的HPLC手性流动相添加剂法,为本品的质量控制提供有效的分析方法。通过优化手性添加剂环糊精种类和浓度、流动相pH和甲醇比例,确定实验条件为Diamonsil C₁₈柱（150 mm×4.6 mm,5 μm）,10 mg/mL磺丁基醚-β-环糊精（SBE-β-CD）溶液（磷酸调pH至2.5）-甲醇（73∶27）为流动相,流速为1.0 mL/ min,柱温40 ℃,检测波长为230 nm。在选定的色谱条件下盐酸度洛西汀对映体之间的分离度为2.3。左旋异构体在1.0~50 μg/mL范围内,峰面积与浓度线性关系良好（r=0.999 9）,左旋异构体平均回收率为100.8 %（RSD=1.0 %）,定量限为0.02 μg。本研究建立的方法简便、准确、专属性强,可用于盐酸度洛西汀中左旋异构体的检查。     

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.     

Extraction and determination of trace amounts of chlorpromazine in biological fluids using magnetic solid phase extraction followed by HPLC

A simple, rapid and sensitive method termed as magnetic solid phase extraction (MSPE) combined with high-performance liquid chromatography-ultraviolet detector (HPLC-UV) has been proposed for the determination of trace amounts of chlorpromazine (CPZ) in water, urine and plasma samples. The separation and determination was performed on a C18 column under the optimal chromatographic conditions. Several factors influencing the extraction efficiency of CPZ, such as pH, surfactant and adsorbent amounts, ionic strength, extraction time, sample volume and desorption conditions, were studied and optimized. Under the optimal MSPE conditions, the extraction percentage of CPZ was 74%, 27% and 16% in water, urine and plasma samples, respectively. The limits of detection (LODs) of the proposed approach were 0.1, 5.0 and 10 ng/mL in water, urine and plasma samples, respectively. The relative standard deviations (RSDs) based on five replicate determinations at 10 ng/mL level of CPZ was 1.2%. Good linear behaviors over the investigated concentration ranges (0.25–300 ng/mL) with good coefficient of determination, R²>0.9998, were obtained. Good spike recoveries with relative errors less than 9.0% were obtained when applying the proposed method to water, urine and plasma samples.     

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.     

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.     

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.     

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.     

Quantitative and qualitative analysis of common peaks in chemical fingerprint of Yuanhu Zhitong tablet by HPLC-DAD–MS/MS

A quality control (QC) strategy for quantitative and qualitative analysis of “common peaks” in chemical fingerprint was proposed to analyze Yuanhu Zhitong tablet (YZT), using high performance liquid chromatography with diode array detector and tandem mass spectrometry (HPLC-DAD–MS/MS). The chromatographic separation was achieved on an Agilent Eclipse plus C₁₈ column with a gradient elution using a mixture of 0.4‰ ammonium acetate aqueous (pH 6.0 adjusted with glacial acetic acid) and acetonitrile. In chemical fingerprint, 40 peaks were assigned as the “common peaks”. For quantification of “common peaks”, the detection wavelength was set at 254 nm, 270 nm, 280 nm and 345 nm, respectively. The method was validated and good results were obtained to simultaneously determine 10 analytes (protopine, jatrorrhizine, coptisine, palmatine, berberine, xanthotoxin, bergapten, tetrahydropalmatine, imperatorin and isoimperatorin). For qualification of “common peaks”, 33 compounds including 10 quantitative analytes were identified or tentatively characterized using LC–MS/MS. These results demonstrated that the present approach may be a powerful and useful tool to tackle the complex quality issue of YZT.     

Simultaneous determination of amlodipine,valsartan and hydrochlorothiazide by LC–ESI-MS/MS and its application to pharmacokinetics in rats

Polypill is a fixed-dose combination that contains three or more active ingredients used as a single daily pill to achieve a large effect in preventing cardiovascular disease with minimal adverse effects. A novel and accurate liquid chromatography tandem mass spectrometry method using electrospray ionization mode has been developed and validated for the simultaneous determination of amlodipine (AMD), valsartan (VAL) using losartan (LOS) as an internal standard (IS), and hydrochlorothiazide (HCT) using furosemide (FSD) as an IS. The separation was carried on Aquasil C₁₈ (50 mm×2.1 mm, 5 µm) reversed phase column using acetonitrile and water containing 0.1% formic acid (50:50, v/v) as the mobile phase. The method was validated in terms of linearity, accuracy and precision over the concentration range of 1–1000 ng/mL. The intra and inter-day precision and accuracy, stability and extraction recoveries of all the analytes were in the acceptable range. This method can be successfully applied to the pharmacokinetic study of AMD, VAL and HCT when given as a polypill.