LC-MS determination of MPTP at sub-ppm level in pethidine hydrochloride

An HPLC-MS with electrospray ionisation method for the determination of MPTP at sub-ppm level in pethidine hydrochloride has been developed and validate. Ionisation is performed by positive-ion electrospray and the quadrupole filter mass spectrometer is operated in the single ion recording mode. Chromatographic separation was achieved in gradient elution using a symmetry C18, 5 μm, 150 mm × 2.1 mm i.d. The mobile phase comprised water containing 0.1% formic acid (v/v) and acetonitrile containing 0.1% formic acid (v/v). The method showed to be linear in the range between 0.2 and 2.2 ng/ml, the estimated LOD was lower than 0.1 ng/ml and the LOQ was lower than 0.2 ng/ml.     

Fast liquid chromatographic-tandem mass spectrometric (LC-MS-MS) determination of metformin in plasma samples

Liquid chromatographic–tandem mass spectrometric (LC–MS–MS) methods for the determination of metformin in plasma from different species are presented. The first method employed a YMC cyano 2 mm×50 mm, 3 μm analytical column. For minimum sample preparation direct injection of samples after protein precipitation was performed. The polar column used with highly organic mobile phases provided a normal phase retention mechanism. The elution conditions were optimized to obtain reproducible peak areas and good peak shape. A step gradient from 100% acetonitrile to acetonitrile–water 80:20 (v/v) containing 10 mM ammonium acetate and 1% acetic acid was applied, leading to a sample-to-sample cycle time of 2 min. In a second method, a column-switching LC–MS–MS assay for on-line trapping was developed. The analyte and internal standard were trapped on a YMC cyano 2 mm×10 mm, 5 μm column using acetonitrile–methanol 95:5 (v/v). Elution was performed isocratically in back-flush mode on to the analytical column (YMC cyano 2 mm×50 mm, 3 μm) using 10 mM ammonium acetate in acetonitrile–water 80:20 (v/v) with 1% formic acid. With this approach, the signal-to-noise ratio was improved and the run time could be shortened to 1 min. Calibration samples were prepared in the matrix to be assayed in the range of 10–10,000 ng/ml. Quality control (QC) samples were prepared at 40, 400 and 4000 ng/ml and interspersed with the unknown study samples in the assays. Deviations for precision and accuracy were less than 20% for the lower limit of quantification (LLOQ) and low QC sample and less than 15% for other calibrators and QCs.     

High-performance liquid chromatographic method for the bioequivalence evaluation of desloratadine fumarate tablets in dogs

A simple HPLC method was developed for the determination of desloratadine in dog plasma and was used for evaluating the bioequivalence of desloratadine fumarate tablets and desloratadine tablets in dogs. Chromatographic separation was performed on a Hypersil CN column (150 mm×5.0 mm, 5 μm) using a mixture of methanol, acetonitrile and phosphate buffer (pH 5.5; 0.01 mol/l) (35:35:30, v/v/v) as mobile phase delivered at a flow rate of 0.8 ml/min. The detection was set at 241 nm. The limit of quantitation was 5.0 ng/ml. The calibration range was from 5.0 to 800.0 ng/ml. Inter- and intra-day precision ranged from 1.8 to 3.8% and from 2.2 to 9.0%, respectively. The recovery of desloratadine from dog plasma ranged from 78.8 to 82.0%. The developed method was applied to the bioequivalence studies of desloratadine fumarate tablets (test preparation) and desloratadine tablets (reference preparation) in five dogs. Pharmacokinetic parameters t_max, C_max, AUC_0–t, AUC_0–∞, t_1/2 were determined from plasma concentration-time profiles of both preparations. The analysis of variance (ANOVA) did not show any significant difference between the two preparations and 90% confidence intervals fell within the acceptable range for bioequivalence. Based on these statistical inferences it was concluded that the two preparations exhibited comparable pharmacokinetic profiles and that desloratadine fumarate tablets was bioequivalent to desloratadine tablets.     

Improved liquid chromatographic method for the simultaneous determination of tramadol and its three main metabolites in human plasma, urine and saliva

Tramadol, an analgesic agent, and its main metabolites O-desmethyltramadol (M1), N-desmethyltramadol (M2) and O,N-didesmethyltramadol (M5) were determined simultaneously in human plasma, saliva and urine by a rapid and specific HPLC method. The sample preparation was a simple, one-step, extraction with ethyl acetate. Chromatographic separation was achieved with a Chromolith™ Performance RP-18e 100 mm × 4.6 mm column, using a mixture of methanol:water (19:81, v/v) adjusted to pH 2.5 by phosphoric acid, in an isocratic mode at flow rate of 2 ml/min. Fluorescence detection (λ_ex 200 nm/λ_em 301 nm) was used. The calibration curves were linear (r² > 0.996) in the concentration ranges in plasma, saliva and urine. The lower limit of quantification was 2.5 ng/ml for all compounds. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were acceptable in all analyzed body fluids The developed procedure was applied to assess the pharmacokinetics of tramadol and its main metabolites following administration of 100 mg single oral dose of tramadol to healthy volunteers.     

Simultaneous determination of roxithromycin and ambroxol hydrochloride in a new tablet formulation by liquid chromatography

A rapid and accurate liquid chromatographic method is described for the simultaneous determination of roxithromycin and ambroxol hydrochloride in a new tablet formulation. Chromatographic separation of the two drugs was achieved on a Diamonsil™ C₁₈ column (200 mm×4.6 mm, 5 μm). The mobile phase consisting of a mixture of acetonitrile, methanol and 0.5% ammonium acetate (39:11:50 (v/v), pH 5.5) was delivered at a flow rate of 1.0 ml/min. Detection was performed at 220 nm. Linearity, accuracy and precision were found to be acceptable over the concentration range of 201.2–2012.0 μg/ml for roxithromycin and 42.7–427.0 μg/ml for ambroxol hydrochloride, respectively. Separation was complete in less than 10 min. The proposed method can be used for the quality control of formulation products.     

Evaluation of impurities level of perindopril tert-butylamine in tablets

Perindopril tert-butylamine is a new member of angiotensin-converting enzyme inhibitors group used in the treatment of hypertension and heart failure. In this paper, the evaluation of reversed-phase high-performance liquid chromatographic method (RP-HPLC) for the determination of impurities level of perindopril tert-butylamine in tablets was done. The chromatograms were recorded using a Hewlett Packard 1100 chromatographic system with DAD detector. Separations were performed on a YMC-Pack C8 column (250 mm × 4.6 mm; 5 μm particle size) at 50 °C column temperature. Mobile phase was a mixture of acetonitrile–potassium phosphate buffer (0.05 M) (37:63, v/v) (pH 2.5). pH of the mobile phase was adjusted with ortophosphoric acid. Mixture of acetonitrile–water (40:60, v/v) was used as a solvent. Injection volume was 50 μl, flow rate 1.7 ml min⁻¹ and UV-detection was performed at 215 nm. The developed method subjected to method validation and parameters in terms of selectivity, linearity, precision, accuracy, limit of detection, limit of quantitation and robustness were defined. The validated method is suitable for the simultaneous determination of perindopril tert-butylamine as well as its impurities in pharmaceuticals.     

Validated enantiospecific LC method for determination of (R)-enantiomer impurity in (S)-efavirenz

A high-performance liquid chromatographic method was developed for separation of the enantiomers of efavirenz. The developed method was applied for the determination of (R)-enantiomer in (S)-efavirenz and satisfactory results were achieved. The base line separation with a resolution of more than 4.0 was achieved on Chiralcel OD (250 mm x 4.6 mm, 10 microm) column containing tris-(3,5-dimethylphenylcarbomate) as stationary phase. The mobile phase consists of n-hexane: isopropyl alcohol (80:20 v/v) with 0.1% (v/v) of formic acid as additive. The flow rate was kept at 1.0 ml/min and the UV detection was monitored at 254 nm. The (R)-enantiomer was found linear over the range of 0.1 microg/ml--6 microg/ml. The limit of detection (LOD) was 0.03 microg/ml and the limit of quantification (LOQ) was 0.1 microg/ml (n=3. The precision of (R)-enantiomer at LOQ level was evaluated through six replicate injections and the RSD of the peak response was achieved as 1.34%. The results demonstrated that the developed LC method was simple, precise, robust and applicable for the purity determination of efavirenz.     

Integrated on-line sample clean-up using cation exchange restricted access sorbent for the LC determination of atropine in human plasma coupled to UV detection

A new, simple and fully automated liquid chromatographic (LC) method with UV detection has been developed for the direct determination of atropine in plasma. Sample clean-up was based on the use of cation exchange restricted access material (RAM) in a pre-column, coupled to LC by means of a column switching system. After direct injection of a 200 μl-volume of plasma sample, the biological matrix was washed out for 10 min using a washing liquid composed of 2 mM lithium perchlorate adjusted to pH 3.0 and methanol (97:3; v/v). By rotation of the switching valve, atropine was then eluted in the back-flush mode for 2 min and transferred to the analytical column packed with octadecyl silica by the LC mobile phase constituted of a mixture of acetonitrile and potassium phosphate buffer (pH 3.0; 50 mM) containing 2 mM sodium heptanesulfonate (16:84; v/v). The UV detection was performed at 220 nm.

The method was validated according to a new approach based on accuracy profile over a concentration range from 25 ng/ml, corresponding to the limit of quantitation, to 1000 ng/ml. The method was then applied for the determination of atropine in plasma after intravenous administration to hospitalised patients.     

A Validated RP-HPLC Method for the Estimation of Lapatinib in Tablet Dosage form using Gemcitabine Hydrochloride as an Internal Standard

A simple, selective, rapid, precise and economical reverse-phase high-performance liquid chromatography method has been developed for the determination of lapatinib in tablet using gemcitabine hydrochloride as an internal standard. Chromatography was carried out on an ODS C-18 RP column (4.6 mm i.d. ×250 mm) using a mixture of acetonitrile and water (50:50 v/v) as the mobile phase at a flow rate of 1.0 ml/min. The drug was monitored at 232 nm. The retention times for lapatinib and gemcitabine hydrochloride were found to be 4.25±0.05 and 6.10±0.05 min, respectively. The method produced linear responses in the concentration range of 2-60 μg/ml of lapatinib. The limit of detection and limit of quantitation were 0.265 and 0.884 μg/ml, respectively.     

Development of a high performance liquid chromatography-tandem mass method for determination of bis(7)-tacrine, a promising anti-Alzheimer's dimer, in rat blood

An analytical method using on-line high performance liquid chromatography-tandem mass spectrometry with electrospray ionization was developed and applied for the quantification of bis(7)-tacrine (B7T) in rat blood. B7T and pimozide (internal standard, IS) were extracted in a single step from 100 μl of alkalized blood with ethyl acetate. Analytes were separated using an Extend C-18 column at 25 °C. The elution was achieved isocratically with a mobile phase composed of 0.05% aqueous formic acid and acetonitrile (60:40, v/v) at a flow rate of 0.35 ml/min. Quantification was achieved by monitoring the selected ions at m/z 247 for B7T and m/z 462 → m/z 328 for pimozide. Retention times were 1.45 and 2.23 min for B7T and IS, respectively. Calibration curves were linear in the range from 86.4 to 2160.0 ng/ml. The established method is rapid, selective and sensitive for the identification and quantification of B7T in biological samples. The assay is accurate (bias <10%) and reproducible (intra- and inter-day variation <10%), with detection and quantification limit of 3.6 and 42.3 ng/ml, respectively. Furthermore, it was successfully applied for the pharmacokinetic measurement of B7T in rat with a single intravenous administration at 0.3 mg/kg.     

Development and validation of a bioanalytical method using automated solid-phase extraction and LC-UV for the simultaneous determination of lumefantrine and its desbutyl metabolite in plasma

A bioanalytical method for the determination of lumefantrine (LF) and its metabolite desbutyl-lumefantrine (DLF) in plasma by solid-phase extraction (SPE) and liquid chromatography has been developed. Plasma proteins were precipitated with acetonitrile:acetic acid (99:1, v/v) containing a DLF analogue internal standard before being loaded onto a octylsilica (3 M Empore) SPE column. Two different DLF analogues were evaluated as internal standards. The compounds were analysed by liquid chromatography UV detection on a SB-CN (250 mm × 4.6 mm) column with a mobile phase containing acetonitrile–sodium phosphate buffer pH (2.0; 0.1 M) (55:45, v/v) and sodium perchlorate 0.05 M. Different SPE columns were evaluated during method development to optimise reproducibility and recovery for LF, DLF and the two different DLF analogues. The within-day precisions for LF were 6.6 and 2.1% at 0.042 and 8.02 μg/mL, respectively, and for DLF 4.5 and 1.5% at 0.039 and 0.777 μg/mL, respectively. The between-day precisions for LF were 12.0 and 2.9% at 0.042 and 8.02 μg/mL, respectively, while for DLF 0.7 and 1.2% at 0.039 and 0.777 μg/mL, respectively. The limit of quantification was 0.024 and 0.021 μg/mL for LF and DLF, respectively. Different amounts of lipids in plasma did not affect the absolute recovery of LF or DLF.     

Determination of endothelin antagonist BMS182874 in plasma by high-performance liquid chromatography

A simple and rapid high performance liquid chromatography (HPLC) method was developed for the determination of BMS182874 (BMS) in mouse plasma. The drug was extracted from plasma by a liquid-liquid extraction process. The method consists of reversed-phase chromatography using a Thermo Hypersil-Keystone RP-18 5 microm, 250 x 2.1 mm column and UV spectrophotometer detection at 255 nm. The mobile phase consists of 45% (v/v) acetonitrile: 55% (v/v) trifluoroacetic acid (0.015% v/v; pH 3.0) at a flow rate of 0.6 ml/min. Validity of the method was studied and the method was precise and accurate with a linearity range from 100 ng/ml to 1000 ng/ml. The extraction efficiency was found to be 81, 84 and 87% for 100, 500 and 1000 ng/ml, respectively for spiked drug in plasma. The limit of quantification and limit of detection were found to be 50 and 10 ng/ml, respectively in plasma. Within-day and between-day precision expressed by relative standard deviation was less than 4% and inaccuracy did not exceed 4%. The assay was also used to analyze samples collected during animal studies. The suitability and robustness of the method for in vivo samples were confirmed by analysis of BMS from mouse plasma and tissues dosed with BMS.     

Analysis of neolignans compounds of Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck by HPLC

A high performance liquid chromatographic (HPLC) method was developed and validated for quantitative determination of neolignans in extracts of Piper regnellii var. pallescens. The analysis were carried out on a Metasil ODS column (150 mm x 4.6 mm, 5 microm) at 30 degrees C, using as mobile phase acetonitrile-water (60:40, v/v) containing 2% acetic acid. The flow rate was 1.0 ml/min and the detection was at 280 nm. The validation using conocarpan as standard demonstrated that the method presents linearity (linear correlation coefficient=0.9991), precision (relative standard deviation <5%) and accuracy (mean recovery=104.55%) in the concentration range 31.25-500 microg/ml. The limit of detection (LOD) was 1.68 microg/ml and the limit of quantitation was 5.60 microg/ml. This method allowed the identification and quantification of conocarpan, eupomatenoid-5 and eupomatenoid-6 in the hydroethanolic extracts obtained from the leaves, stems and roots by maceration process. All the extracts showed the same chromatographic profile, being that the extract of the roots presented the highest concentration of neolignans.     

Validation of a sensitive LC/MS/MS method for simultaneous quantitation of flupentixol and melitracene in human plasma

A sensitive method has been developed and validated, using LC/ESI-MS/MS, for simultaneous quantitation of flupentixol and melitracen—antidepressant drugs, in human plasma. The quantitation of the target compounds was determined in a positive ion mode and multiple reaction monitoring (MRM). The method involved a repeated liquid–liquid extraction with diethyl ether and analytes were chromatographed on a C₈ chromatographic column by elution with acetonitrile–water–formic acid (36:64:1, v/v/v) and analyzed by tandem mass spectrometry. The method was validated over the concentration ranges of 26.1–2090 pg/ml for flupentixol and 0.206–4120 ng/ml for melitracen. The correlation coefficients of both analyst were >0.998 for six sets of calibration curves. The recovery was 60.9–75.1% for flupentixol, melitracen and internal standard. The lower limit of quantitation (LLOQ) detection was 26.1 pg/ml for flupentixol and 0.206 ng/ml for melitracen. Intra- and inter-day precision of the assay at three concentrations were 2.15–5.92% with accuracy of 97.6–103.0% for flupentixol and 0.5–6.36% with accuracy of 98.7–101.7% for melitracen. Stability of compounds was established in a battery of stability studies, i.e., bench-top, autosampler and long-term storage stability as well as freeze/thaw cycles. The method proved to be suitable for bioequivalence study of flupentixol and melitracen in healthy human male volunteers.     

Simultaneous determination of nikethamide and lidocaine in human blood and cerebrospinal fluid by high performance liquid chromatography

Nikethamide and lidocaine are often requested to be quantified simultaneously in forensic toxicological analysis. A simple reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed for their simultaneous determination in human blood and cerebrospinal fluid. The method involves simple protein precipitation sample treatment followed by quantification of analytes using HPLC at 263 nm. Analytes were separated on a 5 μm Zorbax Dikema C₁₈ column (150 mm × 4.60 mm, i.d.) with a mobile phase of 22:78 (v/v) mixture of methanol and a diethylamine–acetic acid buffer, pH 4.0. The mean recoveries were between 69.8 and 94.4% for nikethamide and between 78.9 and 97.2% for lidocaine. Limits of detection (LODs) for nikethamide and lidocaine were 0.008 and 0.16 μg/ml in plasma and 0.007 and 0.14 μg/ml in cerebrospinal fluid, respectively. The mean intra-assay and inter-assay coefficients of variation (CVs) for both analytes were less than 9.2 and 10.8%, respectively. The developed method was applied to blood sample analyses in eight forensic cases, where blood concentrations of lidocaine ranged from 0.68 to 34.4 μg/ml and nikethamide ranged from 1.25 to 106.8 μg/ml. In six cases cerebrospinal fluid analysis was requested. The values ranged from 20.3 to 185.6 μg/ml of lidocaine and 8.0 to 72.4 μg/ml of nikethamide. The method is simple and sensitive enough to be used in toxicological analysis for simultaneous determination of nikethamide and lidocaine in blood and cerebrospinal fluid.     

LC determination of glimepiride and its related impurities

Five impurities in glimepiride drug substance were detected and quantified using a simple isocratic reverse phase HPLC method. For the identification and characterization purpose these impurities were isolated from a crude reaction mixture of glimepiride using a normal phase HPLC system. Based on the spectroscopic data like NMR, FTIR, UV and MS these impurities were characterized and used as impurity standards for determining the relative response factor during the validation of the proposed isocratic reverse phase HPLC method. The chromatographic separation was achieved on a Phenomenex Luna C8 (2) 100 Å, 5 μm, 250 mm × 4.6 mm using a mobile phase consisting of phosphate buffer (pH 7.0)–acetonitrile–tetrahydrofuran (73:18:09, v/v/v) with UV detection at 228 nm and a flow rate of 1 ml/min. The column temperature was maintained at 35 °C through out the analysis. The method has been validated as per international guidelines on method validation and can be used for the routine quality control analysis of glimepiride as active pharmaceutical ingredient (API).     

Development and Validation of a Reversed-Phase High-performance Liquid Chromatography Method for Determination of Desmopressin in Chitosan Nanoparticles

A simple isocratic reversed-phase high performance liquid chromatographic method was developed for determination of released desmopressin from chitosan nanoparticles in the in vitro media. The chromatographic separation was achieved with acetonitrile/water (25:75, v/v), in which water contained 0.1% v/v trifluoroacetic acid with pH=2.5 as mobile phase, a Chromolith^® Performance RP-18e column (150×4.6 mm; 5 μm) kept at 40° and ultraviolet detection at 220 nm. The compound was eluted isocritically at a constant flow rate of 1.6 ml/min. The method was validated according to the International Conference on Harmonisation guidelines. The validation characteristics included accuracy, precision, linearity rang, selectivity, limit of detection, limit of quantitation and robustness. The calibration curve was linear (r>0.9999) over the concentration rang 0.5-100 μg/ml. The limit of detection and limit of quantitation in the release media were 0.05 and 0.5 μg/ml, respectively. The proposed method had an accuracy of and intra- and inter-day precision <4.2. Furthermore, to evaluate the performance of the proposed method, it was used in the analysis of desmopressin level in real samples containing chitosan nanoparticles in the in vitro media.     

Development and validation of a sensitive liquid chromatographic method for the analysis of a novel radioprotectant: ON 01210.Na

ON 01210.Na is a chlorobenzylsulfone derivative with potential property to mitigate the effects of accidental or intentional exposure to life threatening levels of radiation. A simple and sensitive HPLC method was developed and validated for the assay of ON 01210.Na. The isocratic system used a mobile phase consisting of acetonitrile:0.1% trifluroacetic acid in water (60:40, v/v) at a flow rate of 1 ml/min. The method used a C-18 Gemini column (250 mm × 4.6 mm) with column effluents monitored at 254 nm. Forced degradation of the drug was achieved by autoclaving ON 01210.Na with 0.05N HCl, 0.05N NaOH or 1.5% (v/v) hydrogen peroxide. The assay validation parameters evaluated include specificity, linearity, precision, accuracy and sensitivity. The retention time of the drug and the other effluents were well within 7 min. Standard curves were linear over the concentration range of 10–500 μg/ml. The R.S.D. values for the within-day and day-to-day precision ranged from 0.4 to 2.5 and 2.2 to 4.4%, respectively. The R.S.D. for accuracy measurement ranged from 0.85 to 1.7%. The critical level, the detection level and the determination level for this assay were 2.86 ± 0.67, 5.69 ± 0.67 and 15.6 ± 1.8 μg/ml, respectively. A simple, sensitive and stability indicating HPLC assay was developed and validated for the analysis of a novel radioprotectant. This method was used to evaluate the aqueous as well as solid-state stability of this drug during autoclaving.     

Bioanalytical method development and validation of natamycin in rabbit tears and its application to ocular pharmacokinetic studies

A new selective and sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of natamycin in rabbit tears using amphotericin B as internal standard (IS). Chromatographic separation was achieved on a Luna Cyano column (100 mm × 2 mm, 3 μm) using ammonium acetate buffer (pH 4; 3.5mM): methanol (10:90, v/v) as the mobile phase. The run time was 5 min. Detection was performed by negative ion electrospray ionization in multiple reaction monitoring (MRM) mode. The calibration curve was linear over the concentration range from 25 to 800 ng/ml, and lower limit of detection of 12.5 ng/ml. The accuracy and precision of the method were within the acceptable limit of ± 20% at the lower limit of quantitation and ± 15% at other concentrations. Natamycin was stable during the battery of stability studies viz., bench-top, auto-sampler, freeze/thaw cycles and 30 days storage in a freezer at -70 ± 10 °C. The method was successfully applied to the ocular pharmacokinetic studies of natamycin eye drops in New Zealand rabbit tears.     

Method development and validation of repaglinide in human plasma by HPLC and its application in pharmacokinetic studies

In this study, the development and validation of a high-performance liquid chromatography (HPLC) assay for determination of repaglinide concentration in human plasma for pharmacokinetic studies is described. Plasma samples containing repaglinide and an internal standard, indomethacin were extracted with ethylacetate at pH 7.4. The recovery of repaglinide was 92% ± 55.31. Chromatographic separations were performed on Purospher^® STAR C-18 analytical column (4.8 mm × 150 mm; 5 μm particle size). The mobile phase composed of acetonitrile–ammonium formate (pH 2.7; 0.01 M) (60:40, v/v). The flow rate was 1 ml/min. The retention time for repaglinide and indomethacin were approximately 6.2 and 5.3 min, respectively. Calibration curves of repaglinide were linear in the concentration range of 20–200 ng/ml in plasma. The limits of detection and quantification were 10 ng/ml and 20 ng/ml, respectively. The inter-day precision was from 5.21 to 11.84% and the intra-day precision ranged from 3.90 to 6.67%. The inter-day accuracy ranged 89.95 to 105.75% and intra-day accuracy ranged from 92.37 to 104.66%. This method was applied to determine repaglinide concentration in human plasma samples for a pharmacokinetic study.