Simultaneous determination of vincristine, vinblastine, catharanthine, and vindoline in leaves of catharanthus roseus by high-performance liquid chromatography

A simple reversed-phase liquid chromatographic method is developed for the simultaneous quantitation of the anticancerous drugs vincristine, vinblastine, and their precursors catharanthine and vindoline using a Merck Chromolith Performance reversed-phase high-performance liquid chromatography column. A better resolution is obtained in comparison with available particulate-type C18 columns. The column provides good reproducibility and peak symmetry. Chromatography is carried isocratically with a mobile phase of acetonitrile-0.1M phosphate buffer containing 0.5% glacial acetic acid (21:79, v/v; pH 3.5) at a flow rate of 1.2 mL/min and UV detection at 254 nm. Parameters such as linearity, limits of quantitation (LOQ) and detection (LOD), precision, accuracy, recovery, and robustness are studied. The method is selective and linear for alkaloid concentration in the range 0.25 microg-25 microg/mL. The LOQ and LOD are 25, 46, 56, and 32 microg/mL and 8, 14, 18, and 10 microg/mL, respectively. The results of accuracy studies are good. Values for coefficient of variation are 2.50, 1.82, 1.33, and 1.13, respectively. The percent recovery of the alkaloids was found to be 96%, 97%, 98%, and 98%, respectively. Peak purity and homogeneity of these compounds in plant extract is studied using a photodiode-array detector. This simple and rapid method of analysis is applied for the determination of these alkaloids in a large number of leaf extracts of Catharanthus roseus..     

Spectrophotometric determination of Rauwolfia alkaloids: estimation of reserpine in pharmaceuticals.

A simple, sensitive and economically viable spectrophotometric method for the determination of some Rauwolfia alkaloids (ajmaline, ajmalicine, reserpine and yohimbine-HCl) has been developed. The method involves the oxidation of Rauwolfia alkaloids by iron(III) and subsequent complexation of iron(II) with 1,10-phenanthroline, forming a red-colored complex having the maximum absorbance at 510 nm. The method is applied to the determination of reserpine in tablets of pharmaceutical formulations. The common excipients do not interfere with the proposed method. A statistical comparison of these results with those of a reported method shows good agreement and indicates no significant difference in the precision.     

Analysis of fatty oil in Semen Ziziphi Spinosae by capillary gas chromatography

A simple and fast capillary gas chromatographic (CGC) method with flame ionization detection is developed for the analysis of fatty oil in Semen Ziziphi Spinosae. After methyl-esterification, eight components are identified by gas chromatography-mass spectrometry. The derivatization condition is investigated in order to validate this method. Palmitic acid and stearic acid are quantitated simultaneously. The limits of detection are 5.024 microg/mL for palmitic acid and 6.957 microg/mL for stearic acid, respectively. The limits of quantitation are 16.76 microg/mL for palmitic acid and 23.19 microg/mL for stearic acid, respectively. The percent recoveries of palmitic and stearic acid are 97.4% and 96.6%. CGC is shown to be a quick and informative tool for the analysis of fatty oil in Semen Ziziphi Spinosae.     

Simultaneous determination of four lignan compounds in Herpetospermum caudigerum by normal-phase high-performance liquid chromatography

A normal-phase high-performance liquid chromatographic method with diode array UV detection is developed for the simultaneous quantitation of four lignan compounds in Herpetospermum caudigerum. This analysis provides a good resolution and reproducibility. Chromatography is carried out with a mobile phase of N-hexane-dichlormethane-methanol (42.5:42.5:5, v/v) at a flow rate of 1.0 mL/min. UV detection is performed at 280 nm. The calibration curve for lignans concentration is linear over the range of 2.10 to 42.0 microg/mL, 15.26 to 305.2 microg/mL, 6.15 to 123.0 microg/mL, and 6.24 to 124.8 microg/mL, respectively. The limit of quantitation and detection for compounds 1, 2, 3, and 4 is 1.31, 2.74, 2.63, and 2.17 microg/mL and 0.28, 0.25, 0.27, and 0.31 microg/mL, respectively. The validation data show that the assay is sensitive, specific, accurate, and reproducible for the simultaneous quantitation of four compounds. This rapid method is therefore appropriate to quantitate these lignans in Herpetospermum caudigerum.     

Analysis of monoethanolamine by derivatization with Marfey's reagent and HPLC

A sensitive and selective method for determining the residual monoethanolamine in a developmental drug substance is developed and validated. Marfey's reagent, which is commonly used for the chiral analysis of amino acids, is reacted with the primary amine group of monoethanolamine and then analyzed by high-performance liquid chromatography-UV at 340 nm. Quantitation is performed by a standard addition method by preparing drug substance samples with added monoethanolamine ranging from 0.25-1.0 microg/mL (equivalent to 12.5-50 ppm with respect to the drug substance). The method performance is evaluated for linearity, specificity, detection and quantitation limits, accuracy, precision, and sample stability. The method is linear from 0.25-1.0 microg/mL with a coefficient of determination (r(2)) > 0.95. The accuracy and precision obtained is 105.5 +/- 4.8% (n = 3). The limits of detection and quantitation are 0.03 and 0.10 microg/mL, respectively. Instrument precision (% relative standard deviation of six injections of a derivatized 0.5 microg/mL monoethanolamine solution on two separate days) is >/= 2.0%. This method is suitable for the determination of monoethanolamine at the 25 ppm level in drug substance.     

Analysis of yohimbine alkaloid from Pausinystalia yohimbe by non-aqueous capillary electrophoresis and gas chromatography-mass spectrometry

In the present work, the qualitative and quantitative analysis of Pausinystalia yohimbe-type alkaloids in the barks of Rubiaceae species is presented using different analytical approaches. Extracts of P. yohimbe were first examined by GC-MS and the major alkaloids were identified. The quantitation of yohimbine was then accomplished by non-aqueous CE (NACE) with diode array detection. This approach was selected in order to use a running buffer fully compatible with samples in organic solvent. In particular, a mixture of methanol containing ammonium acetate (20 mM) and glacial acetic acid was used as a BGE. The same analytical sample was subjected to GC-MS and NACE analysis; the different selectivity displayed by these techniques allowed different separation profiles that can be useful in phytochemical characterization of the extracts. The linear calibration ranges were all 10-1000 microg/mL for yohimbine by GC-MS and NACE analysis. The recovery of yohimbine was 91.2-94.0% with RSD 1.4-4.3%. The LOD for yohimbine were 0.6 microg/mL by GC-MS and 1.0 microg/mL by NACE, respectively. The GC-MS and NACE methods were successfully validated and applied to the quantitation of yohimbine.     

Rapid method for trace determination of organochlorine pesticides and polychlorinated biphenyls in yogurt

A new multiresidue method was developed for the analysis of 19 organochlorine pesticides and 6 polychlorinated biphenyls in yogurt. The sample was extracted twice with acetone by homogenization with an Ultra-Turrax dispersing unit, and the combined extracts were filtered. The extract was then purified by reversed-phase C18 columns and subjected to further cleanup with neutral alumina columns. The residues were determined by gas chromatography with electron capture detection. After the method was optimized, it was validated by determination of recovery percentages, precision (repeatability and reproducibility), and sensitivity (detection and quantitation limits) with yogurt samples fortified at 10 and 1 microg/kg concentration levels. The recovery of 23 organochlorine residues ranged from 77 to 95% at a level of 10 microg/kg, from 74 to 102% at a level of 1 microg/kg, and between 54 and 61% for dieldrin and alpha-endosulfan. The method is repeatable and reproducible, with relative standard deviation values <19% for all residues except dieldrin. Detection and quantitation limits were between 0.02 and 0.62 microg/kg. The analytical method proposed was quick, accurate, repeatable, and reproducible for the determination of organochlorine residues in yogurt samples.     

Simple, sensitive, selective and validated spectrophotometric methods for the estimation of a biomarker trigonelline from polyherbal gels

Simple, accurate, reproducible, selective, sensitive and cost effective UV-spectrophotometric methods were developed and validated for the estimation of trigonelline in bulk and pharmaceutical formulations. Trigonelline was estimated at 265 nm in deionised water and at 264 nm in phosphate buffer (pH 4.5). Beer's law was obeyed in the concentration ranges of 1-20microg mL(-1) (r2=0.9999) in deionised water and 1-24 microg mL(-1) (r2=0.9999) in the phosphate buffer medium. The apparent molar absorptivity and Sandell's sensitivity coefficient were found to be 4.04 x 10(3)L mol(-1)cm(-1) and 0.0422 microg cm(-2)/0.001A in deionised water; and 3.05 x 10(3)L mol(-1)cm(-1) and 0.0567 microg cm(-2)/0.001A in phosphate buffer media, respectively. These methods were tested and validated for various parameters according to ICH guidelines. The detection and quantitation limits were found to be 0.12 and 0.37 microg mL(-1) in deionised water and 0.13 and 0.40 microg mL(-1) in phosphate buffer medium, respectively. The proposed methods were successfully applied for the determination of trigonelline in pharmaceutical formulations (vaginal tablets and bioadhesive vaginal gels). The results demonstrated that the procedure is accurate, precise, specific and reproducible (percent relative standard deviation <2%), while being simple and less time consuming and hence can be suitably applied for the estimation of trigonelline in different dosage forms and dissolution studies.     

The rapid determination of artemisinin by post-column derivatization high-performance liquid chromatography using matrix solid-phase dispersion method

Artemisinin (an antimalaric compound) is isolated as the active compound of the medicinal plant Artemisia annua L. A simple, rapid, and high-efficient method of extraction is developed, in which it is extracted by matrix solid-phase dispersion (MSPD) and directly analyzed by post-column derivatization high-performance liquid chromatography (HPLC). The quantitation results from the MSPD method are compared with two conventional liquid solvent extraction processes, Soxhlet and ultrasonic wave by F-test, and the result indicates no significant difference. The production rate of Artemisinin during wild plant growth (tested over two years, acquired from Yunnan, provincial Yuan Yan country) is determined. The recovery range of determination is 88.1-91.2%. The relative standard deviation (RSD) is 4.55-6.43%. The limits of detection are 0.1 microg/mL, and the limits of quantitation are 0.5 microg/mL.     

Multiresidue determination of organochlorine pesticides and polychlorinated biphenyls in milk by gas chromatography with electron-capture detection after extraction by matrix solid-phase dispersion

A multiresidue analytical method based on matrix solid-phase dispersion was developed to analyze liquid milk for 22 organochlorine pesticides (OCPs) and 6 polychlorinated biphenyls (PCBs). Initial extraction is performed by loading 3 mL milk onto a 2.0 g octadecyl (C18)-bonded silica cartridge with n-hexane as the eluant. Neutral alumina column chromatography with sodium sulfate as the drying agent is used for further cleanup. The eluate is concentrated to 0.5 mL, and target analytes are determined by capillary gas chromatography with electron-capture detection. The optimized method was validated by determining accuracy (recovery percentages), precision (repeatability and reproducibility), and sensitivity (detection and quantitation limits) from analyses of milk samples fortified at 10 and 1 microg/L levels. Average recoveries were between 74 and 106% for all residues except beta-HCH, beta-endosulfan, and endosulfan sulfate. Both repeatability and reproducibility relative standard deviation values were < 22% for all residues. Detection limits ranged from 0.02 to 0.12 microg/L and quantitation limits were between 0.02 and 0.62 microg/L. The proposed analytical method may be used as a fast and simple procedure in routine determinations of OCPs and PCBs in milk.     

Development of HPTLC-UV absorption densitometry method for the analysis of alprazolam and sertraline in combination and its application in the evaluation of marketed preparations

A new simple, sensitive, and reproducible high-performance thin-layer chromatography method for the estimation of alprazolam and sertraline in combination is developed using silica gel plates with fluorescent indicators. The system is equipped with an automated sample applicator, and the detection was performed at 254 nm by using UV absorption densitometry. The mobile phase consists of carbon tetrachloride, methanol, acetone, and ammonia in the ratio 12:3:5:0.1. The retention factor values for alprazolam and sertraline are found to be 0.52 and 0.70, respectively. The limit of detection of alprazolam and sertraline in the mixture of given proportion is observed to be 0.05 microg/mL and 2.5 microg/mL and the limit of quantitation is 0.2 microg/mL and 10 microg/mL, respectively. The method has shown good linearity in the range of 0.2 microg/mL to 0.65 pg/mL for alprazolam (R2 > 0.9953) and 10 pg/mL to 32.5 microg/mL for sertraline (R2 > 0.9942). The intra- and inter-assay (n=5) variations in the linear range are less than 4% for alprazolam and 6% for sertraline. Three pharmaceutical products containing this combination are analyzed to test the applicability of the new method. The percentage of alprazolam and sertraline in the tablets studied range from 97.7% to 102.82% and 96.5% to 99.9%, respectively.     

Determination of the newer quinolones levofloxacin and moxifloxacin in plasma by high-performance liquid chromatography with fluorescence detection

A simple, accurate, sensitive, and precise reversed-phase (RP) high-performance liquid chromatographic (HPLC) method with fluorescence detection allowing the sensitive and specific quantitation of the newer fluoroquinolones levofloxacin and moxifloxacin is described. Moxifloxacin is used as the internal standard for the determination of levofloxacin and vice versa. A single-step liquid-liquid extraction from human plasma is sufficient for both quinolones. The method is linear from 0.1 to 15 microg/mL and 0.2 to 7 microg/mL for levofloxacin and moxifloxacin, respectively, covering the clinically relevant plasma concentration range. The limits of quantitation are 0.05 microg/mL (levofloxacin) and 0.2 microg/mL (moxifloxacin). The method is successfully applied to plasma drug level monitoring in a volunteer receiving single therapeutic doses of levofloxacin or moxifloxacin at two different occasions.     

Quantitative determination of bioactive alkaloids lysergol and chanoclavine in Ipomoea muricata by reversed-phase high-performance liquid chromatography

A rapid, simple, sensitive, gradient and reproducible, reverse‐phase high‐performance liquid chromatographic method was developed for the quantitative estimation of bioactive alkaloids, lysergol and chanoclavine in the seeds of Ipomoea muricata. The clavine alkaloid, lysergol, is a bioenhancer for the drugs and nutrients. The samples were analyzed by reverse‐phase chromatography on a Waters spherisorb ODS2 column (250 × 4.6 mm, i.d., 10 µm) using binary gradient elution with acetonitrile and 0.01 m phosphate buffer (NaH₂PO₄) containing 0.1% glacial acetic acid at a flow rate of 0.8 mL/min, a column temperature of 25 °C and UV detection at λ 254 nm. The limits of detection (LOD) and quantitation (LOQ) were 0.035 and 0.106 µg/mL for lysergol and 0.039 and 0.118 µg/mL for chanoclavine, respectively. Standard curves were linear in the range of 2–10 µg/mL (r > 99) for both analytes. Good results were achieved with respect to repeatability (RSD < 2%) and recovery (99.20–102.0). The method was validated for linearity, accuracy repeatability, LOQ and LOD. The method is simple, accurate and precise, and may be recommended for routine quality control analysis of I. muricata seed extracts containing these two clavine alkaloids (1, 2) as bioactive principles of the herb. Copyright © 2011 John Wiley & Sons, Ltd.     

Capillary electrophoresis method for fexofenadine hydrochloride in capsules

A simple, accurate, and effective capillary electrophoresis method with ultraviolet absorbance detection was developed and validated for the quantitation of the antihistamine fexofenadine in capsules. The separation was performed with an uncoated fused-silica capillary (47 cm x 75 microm id) and was operated at 20 kV potential. Temperature was maintained at 25 degrees C. The run buffer was prepared with 20mM Na2B4O7 x 10 H2O. Software was used for system control, data acquisition, and analysis. Method validation was performed by evaluation of the analytical parameters linearity, precision, accuracy, limits of detection and quantitation, and specificity. The method was linear (r = 0.9999) at concentrations ranging from 20 to 100 microg/mL, precise (relative standard deviation intra-assay = 1.2, 1.6, and 1.8% and interassay = 1.5%); accurate (recovery = 98.1%); and specific. The limits of detection and quantitation were 0.69 and 2.09 microg/mL, respectively. The method was compared to the liquid chromatography method developed previously by the authors for the same drug, and no significant difference was found between the 2 methods in fexofenadine hydrochloride quantitation.     

Spectrophotometric determination of submicro quantities of Ajmaline

A spectrophotometric method for the determination of 5–100 μg samples of ajmaline is described. Ajmaline gives a red colour with concentrated nitric acid, which is measured at 510 mμ after a definite time interval. The limits of interference from serpentine and ajmalicine are given. The method can be applied directly for the estimation of ajmaline in pharmaceutical preparations.     

A simple high-performance liquid chromatographic method for the estimation of boswellic acids from the market formulations containing Boswellia serrata extract

A simple, rapid, and reproducible reverse-phase high-performance liquid chromatographic method is developed for the estimation of boswellic acids, the active constituents in Boswellia serrata oleo-gum resin. The chromatographic separation is performed using a mobile phase consisting of acetonitrile-water (90:10, % v/v) adjusted to pH 4 with glacial acetic acid on a Kromasil 100 C18 analytical column with flow rate of 2.0 mL/min and detection at 260 nm. The elution times are 4.30 and 7.11 min for 11-keto beta-boswellic acid (11-KBA) and 3-acetyl 11-keto beta-boswellic acid (A-11-KBA), respectively. The calibration curve is linear in the 11.66-58.30 microg/mL and 6.50-32.50 microg/mL range for 11-KBA and A-11-KBA, respectively. The limits of detection are 2.33 microg/mL and 1.30 microg/mL for 11-KBA and A-11-KBA, respectively. The mean recoveries are 98.24% to 104.17% and 94.12% to 105.92% for 11-KBA and A-11-KBA, respectively. The inter- and intra-day variation coefficients are less than 5%. The present method is successfully applied for the estimation of boswellic acids from the market formulations containing Boswellia serrata extract.     

High-performance liquid chromatography assays for desmethoxyyangonin, methysticin, kavain and their microsomal metabolites

Three novel, simple and reproducible high-performance liquid chromatography quantitative assays with UV detection were developed and validated for three major kavalactones--desmethoxyyangonin, methysticin and kavain--in rat liver microsomes using diazepam as an internal standard; liquid-liquid extraction was used for sample preparation and analysis was performed on a Shimadzu 10A high-performance liquid chromatography system. The analysis was carried out in reversed-phase mode with a Luna C(18) column (150 x 2.00 mm, 3 microm) at 40 degrees C. The limit of quantitation was 0.1 microg/mL using 0.25 mL of microsomal solution. The assays were linear over the range 0.1-10 microg/mL for desmethoxyyangonin, methysticin and kavain. Quality control samples exhibited good accuracy and precision with relative standard deviations lower than 15% and recoveries between 85 and 105%. The assays exhibited satisfactory performance with high sensitivity for quantifying desmethoxyyangonin, methysticin and kavain in rat liver microsomes and were successfully used to determine the three kavalactones and their microsomal metabolites.     

Validation assay for total flavonoids, as rutin equivalents, from Trichilia catigua Adr. Juss (Meliaceae) and Ptychopetalum olacoides Bentham (Olacaceae) commercial extract

An ultraviolet spectrophotometric method was validated for total flavonoid quantitation, as rutin equivalents, present in the Trichilia catigua Adr. Juss (Meliaceae) and Ptychopetalum olacoides Bentham (Olacaceae) commercial extract. Parameters as linearity, interval (range), specificity, estimated limit of detection (LOD, microg/mL), estimated limit of quantitation (LOQ, microg/mL), recovery (R, %), precision or relative standard deviation (RSD, %), and accuracy (E, %) were established. The analytical method was validated according to the experimental results: correlation coefficient (r = 0.9997); interval (RSD = 0.15-0.47%; E = 98.98-101.24%); specificity to total flavonoids quantitation, as rutin equivalents, at wavelength 361.0 nm; LOD = 0.09 microg/mL and LOQ = 0.27 microg/mL; R = 99.36-102.14%; adequate intra- and interrun precision (0.30-0.49% and 0.31-0.81%), and intra- and interrun accuracy (100.60-102.38% and 98.58-100.38%).     

Simultaneous quantification of some pharmaceutical Catharanthus roseus leaf and root terpenoid indole alkaloids and their precursors in single runs by reversed-phase liquid chromatography

A new, rapid, sensitive, and reproducible reversed-phase liquid chromatographic (LC) method with photodiode array detection is described. It allows, in a single run of 30 min, simultaneous separation of 6 pharmaceutically and biologically important Catharanthus roseus leaf and root terpenoid indole alkaloids (TIAs) and 3 of their precursors: TIA precursors tryptophan, tryptamine, and loganine; and TIAs serpentine, catharanthine, ajmalicine, vincristine, vinblastine, and vindoline. The method involves the use of a Phenomenex Luna 5 microm, C18 column (250 mm x 4.6 mm id) and a linear binary gradient mobile phase profile. Detection is performed at 220 and 254 nm, which provided good absorptivity for all of the roseus compounds listed above and gave a minimum detection limit of 0.02 microg/mL. The extraction efficiency, peak purity, and homogeneity parameters of the profiles could be validated using a photodiode array detector. The method was successfully used to quantify major components of leaf and root extracts of C. roseus accessions. The new method thus provides a reliable tool for rapid screening of C. roseus samples in large numbers, which is needed in breeding/genetic engineering and genetic mapping experiments and for monitoring the reaction products, in the in vitro/in vivo conversions of precursors into products, and vice versa.     

Determination of Danshensu, a major active compound of Radix Salvia miltiorrhiza in dog plasma by HPLC with fluorescence detection.

A simple and sensitive high performance liquid chromatography method has been developed for the determination of Danshensu (3, 4-dihydroxyphenyllactic acid) in dog plasma. Plasma samples were extracted with ethyl acetate. Analysis of the extracts was performed on a reversed-phase column with an aqueous phosphate buffer-acetonitrile (93:7, v/v) mobile phase, and 4-hydroxybenzoic acid was used as the internal standard. Fluorescence detection at 285 nm (excitation) and 320 nm (emission) was employed. Standard curves were linear in the range from 0.125 to 11.3 microg/mL (regression coefficient r > 0.993) on three different days. Mean recovery was determined as 96.4% by analysis of plasma standard containing 0.63, 5.65 and 11.3 microg/mL of Danshensu. The inter-day precision (RSD) ranged from 3.4 to 8.6% at concentrations of 0.125, 1.88, 6.28 and 11.3 microg/mL, and the intra-day precision was better than 7.2%. The detection and quantitation limits were 0.063 and 0.125 microg/mL, respectively. This validated assay was applied to the determination of Danshensu concentration in dog plasma after oral administration of Radix Salvia miltiorrhiza extracts.