A validated liquid chromatographic method for estimation of solasodine in various Solanum species and market formulations

This paper describes an improved liquid chromatographic (LC) method involving a prechromatographic derivatization step for the estimation of solasodine from berries of various Solanum species, market samples of Solanum xanthocarpum herb, extract, and its market formulations. Solasodine has heterocyclic nitrogen but has no conjugated double bonds in its structure. However, in all reported LC methods, detection was made in the ultraviolet range of 200-213 nm. In the present study, a prechromatographic derivatization of solasodine was done by forming an ion-pair complex of the heterocyclic nitrogen using the acidic dye methyl orange and acetate buffer of pH 4.7. Detection could be made in the visible range at 530 nm in this method. The method was validated and successfully applied to determine solasodine content in various plant samples and polyherbal formulations. The relative standard deviation was found to be 0.025% for system precision, and 0.8% for the linearity of the method, and the correlation coefficient was 0.999. Plant samples and market formulations were analyzed and found to contain solasodine in the range of 0.113-0.227% (w/w) on a fresh weight basis in berries; 0.3-1.278% (w/w) and 0.412% (w/w) on a dry weight basis in S. xanthocarpum herb powder and extract, respectively; and 0.245-0.525% (w/w) on dry weight basis in formulations containing S. xanthocarpum herb powder. No matrix interference was encountered. The method was found to be accurate, with a mean recovery of 100.5 +/- 0.83%. The method has good reproducibility and was found to be suitable for estimation of solasodine.     

A reversed-phase high-performance liquid chromatographic method for the determination of zafirlukast in pharmaceutical formulations and human plasma

Zafirlukast (ZAF) is a leukotriene receptor antagonist used in the treatment of chronic asthma. In this study, a simple and sensitive reversed-phase, high-performance liquid chromatographic method was developed for the determination of ZAF in pharmaceutical formulations and human plasma. Piribedil was used as an internal standard. Analysis was carried out on a Nucleosil C18 100 A (150 mm x 4.6 mm id, 5 Vm) column with acetonitrile-pH 3.0 acetate buffer (70 + 30, v/v) as the mobile phase at a flow rate of 0.8 mL/min. The peak was detected by an ultraviolet detector set at a wavelength of 240 nm. The retention times were about 3.9 min for piribedil and 5.8 min for ZAF. The developed method was applied to the determination of ZAF in its pharmaceutical formulation and spiked human plasma. For quantification of ZAF in spiked plasma, proteins were precipitated with ethanol before chromatographic analysis. The calibration range was linear from 49.69-437.50 ng/mL in spiked plasma. The absolute recovery from spiked plasma was 98.73 +/- 0.42% at a concentration of 254.78 ng/mL of ZAF. No endogenous substances from plasma were found to interfere.     

A high-performance liquid chromatographic method for the determination of monofluoroacetate

A simple isocratic high-performance liquid chromatographic (HPLC) method for the quantitative analysis of monofluoroacetic acid (MFA), the toxic substance of Dichapetalum cymosum, in plant material, rumen contents (gastric contents), and liver samples is described. A suitable HPLC column that gives optimum sensitivity, accuracy, precision, and separation of MFA is identified. A C-610 organic acid analysis column at ambient temperature with 0.02M H3PO4 as an eluent and ultraviolet detection at 210 nm is utilized to quantitate MFA. Using this method, the average percentage recovery in plant material, bovine liver, and rumen samples is 94.8%, and a detection limit of 12 microg/L is achievable.     

Method development and validation of a high-performance liquid chromatographic method for pyrethrum extract.

A robust analytical methodology is developed for the quantitative determination of all six insecticidal components of pyrethrum extract: jasmolin I, cinerin I, pyrethrin I, jasmolin II, cinerin II, and pyrethrin II. This method, based on the separation technique normal-phase high-performance liquid chromatography, offers selectivity, accuracy, precision, linearity, range, ruggedness, and robustness as well as efficiency and ease.     

A fast and simple high-performance liquid chromatographic assay for aryl hydrocarbon hydroxylase

A simple high-performance liquid chromatographic method using fluorescence detection of the remaining substrate is described for the determination of benzo[alpha]pyrene hydroxylase activity. This assay is far simpler than the previous ones, as it does not require extraction or centrifugation and the measurement occurs directly after dilution of the total incubation medium. The aryl hydrocarbon hydroxylase (AHH) activities in rat liver microsomes are in agreement with those obtained by radioactive assays. Moreover, this assay allows the routine determination of the AHH activity in animal tissues.     

Simple and low-cost high-performance liquid chromatographic method for determination of D- and L-amino acids

In this article, a simple and low-cost method for the analysis of amino acid enantiomers by using high-performance liquid chromatography (HPLC) is described. In this method, the amino acids are modified to diastereomers in order to be separated into enantiomers on a usual C(18) reversed-phase column. Methanol instead of acetonitrile is used as an elution solvent; the results of HPLC with methanol elution are comparable with those of HPLC with acetonitrile elution. Sub-nanomolar sensitivity is attained by measuring the absorbance at 340 nm in analysis of 15 amino-acid enantiomers.     

Direct high-performance liquid chromatographic method for enantioselective and diastereoselective determination of selected pyrethroic acids

This study reports on the direct HPLC stereoisomer separation of selected pyrethroic acids employing commercial cinchona alkaloid derived chiral stationary phases (CSPs). cis/trans-Chrysanthemic acid (cis/trans-CA), cis/trans-chrysanthemum dicarboxylic acid (cis/trans-CDCA), cis/trans-permethrinic acid (cis/trans-PA), and fenvaleric acid (FA) were resolved into the individual stereoisomers, i.e. enantiomers and diastereomers as well. To achieve satisfactory baseline separation an optimisation of the variables of the chromatographic method including chemical structure of the cinchona carbamate CSP, mobile phase composition, and flow rate was required. All four stereoisomers of PA were successfully separated in a single run (alphacis = 1.20, alphatrans = 1.26, critical Rs = 1.65) with an acetonitrile (ACN)-based polar-organic eluent. The complete baseline resolution of all CA stereoisomers succeeded in polar-organic (alphacis = 1.20, alphatrans = 1.35, critical Rs = 3.03) as well as in acetonitrile-based reversed-phase media (alphacis = 1.24, alphatrans = 1.22, critical Rs = 2.73). The latter elution mode was also found to be suitable for the enantio- as well as diastereoselective resolution of CDCA (alphacis = 1.09, alphatrans = 1.50, critical Rs = 1.43), which is to the best of our knowledge the first reported enantiomer separation of this analyte. The enantiomers of FA could be baseline separated employing also reversed-phase mode (alpha = 1.16, Rs = 2.91). These separation methods may be applied for quality control processes in the production of stereoisomerically pure insecticides as well as stereoselective toxicokinetic studies, as CDCA, PA, and FA are suitable biomarkers for monitoring human pyrethroid burden.     

Reversed-phase high-performance liquid chromatographic method for the assay of oxytetracycline

The British Pharmacopoeia monograph for oxytetracycline calcium describes an high-performance liquid chromatographic (HPLC) assay which requires packing of the column by the analyst. Presented in this report is an HPLC method for the assay of oxytetracycline which employs a commercially available reversed-phase column and a solvent system which gives improved separation of the antibiotic from common impurities. Results obtained using this method for both bulk and dosage forms of oxytetracycline are in accord with the results of the microbiological assays.     

A high-performance liquid chromatographic method for determination of the niguldipine analogue DHP-014

A simple and reliable reversed-phase high-performance liquid chromatography method was developed and validated for the determination of DHP-014, a niguldipine analogue with potent P-glycoprotein inhibitory and negligible calcium channel blocking properties, in rat plasma. DHP-014 and niguldipine hydrochloride (the internal standard) were extracted from rat plasma by liquid extraction using hexane. DHP-014 was then separated by HPLC on a C18 column and quantified by ultraviolet detection at 238 nm. The mobile phase consisted of acetonitrile-aqueous 5 mM phosphate buffer (65:35, v/v) containing 0.4% (v/v) triethylamine adjusted to pH 7.0. The mean extraction efficiency of DHP-014 was 109.0 +/- 12.9, 97.7 +/- 8.0 and 102.9 +/- 7.5% for DHP-014 concentrations of 10, 50 and 100 nM, respectively (n = 5). The method was linear over the concentration range 2.5-200 nM with a regression coefficient of 0.998. The limit of detection of DHP-014 in rat plasma was 1.0 nM. The intra- and inter-day coefficients of variation for DHP-014 in rat plasma were 4.7-7.9 and 6.9-9.9%, respectively. The intra- and inter-day accuracy was 98.2-99.5 and 97.9-103%, respectively. The bioanalytical technique was used to determine DHP-014 in plasma samples in a pharmacokinetic study of DHP-014 administered to female Sprague-Dawley rats.     

Rapid high-performance liquid chromatographic method for the determination of peroxyacetic acid

The selective oxidation of methyl p-tolyl sulfide (MTS) to the corresponding sulfoxide (MTSO) by peroxyacetic acid and the subsequent rapid separation of the sulfide and sulfoxide are the basis for a fast and reliable HPLC method for the determination of this oxidizing agent in the presence of hydrogen peroxide. The time required for chromatographic separation was reduced to less than 1 min. To improve the long-term stability of the sulfoxide solution, hydrogen peroxide was decomposed catalytically by manganese dioxide. Even in the presence of a tenfold molar excess of hydrogen peroxide, a storability of at least 20 h without a significant increase in MTSO concentration was achieved. External calibration can be performed using the stable and commercially available MTSO. Real samples from a brewery cleaning-in-place disinfection process were analysed and the results were compared with those of the classical two-step titration.     

Development of a simple and sensitive high-performance liquid chromatography method for determination of glucosamine in pharmaceutical formulations

A column high-performance liquid chromatography (HPLC) method was developed for the determination of glucosamine in dosage forms. Glucosamine was derivatized by addition of a solution containing orthophthaldialdehyde. The HPLC separation was achieved on a Spherimage 80 ODS2 column (250 x 4 mm id, 5 microm particle size) using an isocratic mobile phase containing phosphate buffer-methanol (90 + 10, v/v, pH 6.50) and methanol-tetrahydrofuran (97 + 3, v/v) in proportions of 85 + 15 at a flow rate of 1 mL/min, followed by fluorescence detection. The method was validated for specificity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). The detector response for glucosamine HCI was linear over the concentration range of 0.1-20 microg/mL with a correlation coefficient of 0.9980. The accuracy was between 99.4 and 100.8%. The LOD and the LOQ were 0.009 and 0.027 microg/mL, respectively. The method was applied to determination of glucosamine in solid dosage forms.     

A high-performance liquid chromatographic method for saikosaponin a quantification in rat plasma

A high-performance liquid chromatographic method with UV detection has been developed for the determination of saikosaponin a in rat plasma. Saikosaponin a and internal standard jujuboside A were isolated from plasma samples by solid-phase extraction. The chromatographic separation was achieved on a reversed-phase C(18) column with the mobile phase of acetonitrile-water (35:65, v/v) at a flow rate of 1 mL/min and UV detection was set at 205 nm. The standard curve for saikosaponin a was linear over the concentration range 0.25-10 microg/mL and the limit of detection was 0.05 microg/mL. The absolute recovery was greater than 82%. The precision and accuracy ranged from 3.05 to 9.59% and 95.61 to 110.00%, respectively. The validated method was used to determine saikosaponin a in plasma samples in a pharmacokinetic study of saikosaponin a administered to Sprague-Dawley rats.     

A reversed-phase high-performance liquid chromatographic method to analyze retinal isomers

A high-performance liquid chromatographic (HPLC) procedure was developed to separate all-trans-, 13-cis-, 11-cis- and 9-cis-retinal isomers. Two reversed-phase Vydac C18 columns in series were used with an isocratic solvent system of 0.1 M ammonium acetate-acetonitrile (40:60, v/v) as mobile phase and all-trans-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-no natetraene-1-ol (TMMP) as internal standard. Prior to HPLC, the retinal isomers were efficiently extracted in their original isomeric conformation using dichloromethane-n-hexane in the presence of formaldehyde. This technique is suitable for the assay of 11-cis- and all-trans-retinal isomers in retina.     

Comparative pharmacokinetic study of two boswellic acids in normal and arthritic rat plasma after oral administration of Boswellia serrata extract or Huo Luo Xiao Ling Dan by LC‐MS

Huo Luo Xiao Ling Dan (HLXLD), a Chinese herbal formula composed of 11 different herbs, has been used traditionally for the treatment of arthritis and other chronic inflammatory diseases. However, the pharmacokinetic profile of its anti‐inflammatory bioactive compounds has not been elucidated. Boswellic acids are the bioactive compounds with potent anti‐inflammatory activity isolated from Boswellia serrate which is one of the 11 herbs of HLXLD. The objective of the study was to compare the pharmacokinetics of the two bioactive bowsellic acids: 11‐keto‐β‐boswellic acid and 3‐O‐acetyl‐11‐keto‐β‐boswellic following oral administration of HLXLD or Boswellia serrata extract alone in normal and arthritic rats. An LC‐MS method was developed and validated for the determination of 11‐keto‐β‐boswellic acid and 3‐O‐acetyl‐11‐keto‐β‐boswellic in the comparative pharmacokinetic study. The results showed that there were significant differences in pharmacokinetic parameters between normal and arthritic groups. Interestingly, the absorptions of two boswellic acids were significantly higher in HLXLD than Boswellia serrata extract alone, indicating the synergistic effect of other herbal ingredients in HLXLD. This comparative pharmacokinetic study provided direct evidence supporting the notion that the efficacy of a complex mixture such as HLXLD is better than that of single components in treating human diseases. Copyright © 2014 John Wiley & Sons, Ltd.     

A direct high-performance liquid chromatographic method for the determination of therminol 66 contamination in commercial glycerin and fatty acids.

A simple and sensitive high-performance liquid chromatographic method for the determination of Therminol 66 thermal heating fluid in glycerin and fatty acids is developed. Sample solutions dissolved in methanol-tetrahydrofuran (50:50, v/v) are injected directly into a reversed-phase C18 column and eluted with a methanol and water mixture (88:12, v/v). The concentration of the thermal heating fluid is monitored by fluorescence detection at 257 nm (excitation) and 320 nm (emission). The calibration graph obtained from various concentrations of the thermal heating fluid in the methanol and tetrahydrofuran mixture is linear (correlation coefficient = 0.999), and the limit of detection is 0.01 microg/mL. Spiked glycerin containing 0.1 to 1.0 microg/g of the thermal heating fluid also gives good linearity with a mean recovery of 95.3%. The mean intra- and interassay precision are 1.80-6.51% and 5.71-9.03%, respectively, at the 0.1-microg/g level. The method is simple and does not require any pretreatment step, thus it is ideal for quality assurance purposes.     

Direct and indirect high-performance liquid chromatographic enantioseparation of beta-amino acids

Direct and indirect reversed-phase (RP) high-performance liquid chromatographic methods were developed for the separation of enantiomers of 18 unnatural beta-amino acids, including several beta-3-homo amino acids. The direct separations of the underivatized analytes were performed on chiral stationary phases (CSPs) containing macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T column) and teicoplanin aglycone (Chirobiotic TAG column). The indirect method involved pre-column derivatization with a new chiral derivatizing agent (CDA), (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester ((S)-NIFE), and subsequent separation of diastereomers on Discovery C18 and Hyperpep 300 C18 columns. The different methods were compared in systematic chromatographic examinations. The effects of organic modifier, mobile phase composition, pH and flow rate on the separation were investigated.     

A validated quantitative thin-layer chromatographic method for estimation of diosgenin in various plant samples, extract, and market formulation

A sensitive and reproducible thin-layer chromatographic method has been developed for quantitation of diosgenin, a spiroketal sapogenin. The spots were visualized by spraying with modified anisaldehyde-sulfuric acid reagent. The concentration of anisaldehyde was reduced to 0.1% instead of 1%, and the concentration of sulfuric acid was kept at a minimum of 2%. This successfully reduced charring and background interference. The method was validated according to International Conference on Harmonization guidelines. The method was used for determination of diosgenin from dried samples of fenugreek seeds, leaves, stem, seed extracts, and a polyherbal antidiabetic formulation containing fenugreek powder as one of the ingredients. Increased detection sensitivity was observed with linearity from 98 to 588 ng/spot and a correlation coefficient (r2) of 0.988. The relative standard deviation value for linearity of the method was found to be 0.18%. The method was successfully applied to various plant samples of fenugreek (Methi) with a recovery of 98.11 +/- 1.4%. Dried plant samples and a market formulation were analyzed and found to contain diosgenin in the range of 0.529-0.658% (w/w) in fenugreek seed powders, 0.087% (w/w) in fenugreek leaf powder, 0.015 and 1.27% (w/w) in fenugreek stem powder and extract, respectively, and 0.586% (w/w) in a formulation containing fenugreek seed powder. No matrix interference was observed.