Simultaneous estimation of hydroxychavicol and chlorogenic acid from Piper betel L. through RP-HPLC

A RP-HPLC method was developed (λ (max)?=?280) to quantify hydroxychavicol and chlorogenic acid in Piper betel Linn. The method was validated for linearity, limit of detection (LOD?=?3:1σ/S), limit of quantification (LOQ?=?10:1σ/S), precision, accuracy and ruggedness. The response was linear with good correlation between concentration and mean peak area through a coefficient of determinants (r (2)) of 0.9940, y?=?1.98e?+?004x?+?5.19e?+?004 and 0.9945, y?=?2.76e?+?004x?+?1.40e?+?005 with LOD 1.6?μg?mL(-1), 1.0?μg?mL(-1) and LOQ 5.0?μg?mL(-1) and 3.0?μg?mL(-1), respectively, for hydroxychavicol (28.56% w/w) and chlorogenic acid (0.40% w/w). The %RSD of precision and recovery of hydroxychavicol and chlorogenic acid were <2.0%. The proposed method was simple, accurate, specific, precise and reproducible.     

High-performance liquid chromatographic analysis of amino acids in ackee fruit with emphasis on the toxic amino acid hypoglycin A

High-performance liquid chromatography is used to determine the amino acid content of ripe and unripe ackee fruit. Specific emphasis is placed on the level of the toxic amino acid hypoglycin A (hyp-A) in the unripe and ripe ackee fruit and seed. Unripe samples are found to contain significantly higher quantities (P < 0.05) of hyp-A when compared with ripe samples. Uncooked unripe fruit is found to contain 124.4 +/- 6.7 mg/100 g fresh weight and uncooked ripe fruit 6.4 +/- 1.1 mg/100 g fresh weight. The seed of the uncooked unripe fruit is found to contain 142.8 +/- 8.8 mg/100 g fresh weight, and the seed of uncooked ripe fruit has 106.0 +/- 5.4 mg/100 g fresh weight. Boiling fruit in water for approximately 30 min is efficient in removing hyp-A from the edible arilli; however, low levels of 0.54 +/- 0.15 mg/200 mL are detected in the water that was used to cook the ripe fruit. The average %recovery of the amino acids was 80.34%.     

Development and validation of a solid phase micro-extraction-gas chromatography-mass spectrometry method for the determination of furan in baby-food

An efficient and simple method for the determination of furan in baby-food (vegetables and fruits) by solid phase micro-extraction-gas chromatography-mass spectrometry (SPME-GC-MS) was developed and validated. Experimental design was used to investigate the effects of temperature and time of extraction. The calculated regression model was used to find the experimental conditions providing the optimal SPME extraction yield. Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values in the low ngkg(-1) were achieved, whereas linearity was established over two order of magnitude. Good precision was obtained both in terms of intra-day repeatability and between-day precision on two concentration levels (RSD% lower than 3.6%). Recovery values of 91.5+/-6.2% and of 96.1+/-1.3% (n=3) were calculated at 75 ngkg(-1) and 75 microgkg(-1) level. Finally, the applicability of the method to the determination of furan in a number of commercial and home-made baby-food samples was demonstrated.     

A reversed-phase ultra-fast liquid chromatography–photodiode array detector (RP-UFLC-PDA) method for simultaneous estimation of ayapanin and umbelliferone in Ayapana triplinervis Vahl

A rapid validated ultra-fast liquid chromatography–photodiode array detector (UFLC-PDA) method was developed to identify and quantify ayapanin (AY) and umbelliferone (UM) simultaneously in Ayapana triplinervis Vahl methanolic extract. The method was validated for linearity, limit of detection (LOD; 3:1σ/S), limit of quantification (LOQ; 10:1σ/S), precision, accuracy, specificity and robustness. The response was linear with a good correlation between concentration and mean peak area through a correlation coefficient of 0.9996, y = 7025.7x – 2269.8 and 0.9997, y = y = 16,262x – 946 with LOD of 6.256 ± 0.52 and 3.325 ± 0.36, and LOQ of 18.838 ± 0.18 and 8.870 ± 0.85 for AY (0.67% w/w) and UM (0.18% w/w), respectively. The relative standard deviation (%) of precision and recovery of AY and UM was <2.0%. The proposed method was simple, accurate, specific, precise and reproducible.     

Determination of folic acid in tablets by microemulsion electrokinetic chromatography

A microemulsion electrokinetic chromatography (MEEKC) method has been developed and validated for the determination of folic acid, a water-soluble vitamin, in a commercial tablet formulation. The analysis was performed using a microemulsion containing 0.5% (w/w) ethyl acetate, 1.2% (w/w) butan-1-ol, 0.6% (w/w) sodium dodecyl sulfate, 15% (v/v) 2-propanol and 82.7% (w/w) 10 mmol L(-1) sodium tetraborate aqueous buffer at pH 9.2. Direct UV detection at 214nm led to an adequate sensitivity without interference from sample excipients. For quantitative purposes, niacin was used as internal standard. Acceptable precision (<1.2% relative standard deviation (R.S.D.)), linearity (r = 0.9992; range from 160.0 to 240.0 microg/mL), sensitivity (limit of detection (LOD) = 2.98 microg/mL; limit of quantification (LOQ) = 9.05 microg/mL) and recovery (99.8 +/- 1.8% at three concentration levels) were obtained. Based on the performance characteristics, the proposed methodology was found suitable for the determination of folic acid in tablet formulations.     

Development and validation of gas chromatography-mass spectroscopy method for determination of prilocaine HCl in human plasma using internal standard methodology

The accurate determination of prilocaine HCl levels in plasma is important in both clinical and pharmacological/toxicological studies. Prilocaine HCl is quickly hydrolyzed to o-toluidine, causing methemoglobinemia. For this, the present work describes the methodology and validation of a GC-MS assay for determination of prilocaine HCl with lidocaine HCl as internal standard in plasma. The validation parameters of linearity, precision, accuracy, recovery, specificity, limit of detection and limit of quantification were studied. The range of quantification for the GC-MS was 20-250 ng/mL in plasma. Within-day and between-day precision, expressed as the relative standard deviation (RSD) were less than 6.0%, and accuracy (relative error) was better than 9.0% (n = 6). The analytical recovery of prilocaine HCl and IS from plasma has averaged 94.79 and 96.8%, respectively. LOQ and LOD values for plasma were found to be 20 and 10 ng/mL, respectively. The GC-MS method can be used for determination from plasma of prilocaine HCl in routine measurement as well as in pharmacokinetic studies for clinical use.     

Development and validation of a solid-phase extraction and gas chromatography-tandem mass spectrometry method for the determination of isopropyl-9H-thioxanthen-9-one in carton packaged milk

A simple and efficient method for the determination of isopropyl-9H-thioxanten-9-one (ITX) in different fat content milk samples and baby milk samples stored in packaged cartons was developed and validated. Samples were extracted using solid-phase extraction (SPE) and analysed by gas chromatography-tandem mass spectrometry operated in selected reaction monitoring mode (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values in the low microg/L were achieved, whereas linearity was established within 0.5-500 microg/L range. Good precision was obtained both in terms of intra-day repeatability and inter-day precision on two concentration levels (RSD% lower than 2%). Good percentage recoveries were obtained (92.0-102.0%) even in the presence of high amount of fat. Finally, the developed method was successfully applied to analyse a number of commercial milk samples with different fat content and baby milk samples.     

Bioremediation of toluene and naphthalene: development and validation of a GC-FID method for their monitoring

Bioremediation of toluene and naphthalene in liquid cultures of bacteria grown in the presence of these aromatic compounds as unique sources of carbon was investigated by gas chromatography (GC). For this purpose, a method based on the use of GC with flame ionization detection was developed and validated. Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. In the case of naphthalene, LOD and LOQ values of 0.43 and 0.72 mg kg(-1) were achieved. Linearity was established over one order of magnitude in the range of interest, i.e. 10-100 mg kg(-1). Excellent precision was obtained both in terms of intra-day repeatability and between-day precision on two concentration levels (RSD% lower than 0.5%). A recovery of 97.9 +/- 0.2% (n=3) was calculated by addition of 640 mg kg(-1) of naphthalene to the Bushnell & Haas mineral salts basal solution containing the micro-organisms. Findings clearly showed a reduction of the naphthalene content equal to 50% and 75% after two and four weeks of contact with the micro-organisms, whereas a lower degradation was shown in the case of toluene. Finally bioremediation activity was ascribed to two different microbial populations, Bordetella Petrii and Bacillus Sphericus, which survived in the polluted medium.     

Determination of spinosad at trace levels in bee pollen and beeswax with solid–liquid extraction and LC–ESI‐MS

This paper reports the use of a new LC method with a fused‐core analytical column coupled to ESI‐MS to determine residues of the biopesticide spinosad in bee pollen and beeswax. The method analyzes the active ingredients, spinosyns A and D, with a simple and efficient sample treatment (recovery between 90 and 105%) consisting of a solid–liquid extraction with acetone (bee pollen) or acetonitrile (beeswax). The method was validated in terms of selectivity, LOD, LOQ, linearity, and precision. The LOD and LOQ values ranged between 0.1–0.2 and 0.4–0.7 μg/kg, respectively. Moreover, the precision obtained within the linear concentration range (LOQ 500 μg/kg) was satisfactory (RSD lower than 5%). Finally, the proposed method was applied to analyze bee pollen and beeswax samples collected from apiaries located close to fruit orchards in two Spanish regions.     

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%).     

UV spectrophotometric determination of bioflavonoids from a semisolid pharmaceutical dosage form containing Trichilia catigua Adr. Juss and Ptychopetalum olacoides Bentham standardized extract: analytical method validation and statistical procedures

A precise, accurate, and sensitive UV spectrophotometric method was developed and validated for routine quantification of total bioflavonoids, expressed as rutin, from a topical oil-in-water pharmaceutical emulsion containing the extract of Trichilia catigua Adr. Juss and Ptychopetalum olacoides Bentham. The method was validated experimentally, and the data were treated rigorously by statistical analysis. The following analytical parameters were assessed: linearity, specificity, intra- and interrun precision measured as relative standard deviation (RSD, %), intra- and interrun accuracy (E, %), recovery (Rec., %), limit of detection (LOD, microg/mL), and limit of quantification (LOQ, microg/mL). The UV spectrophotometric method was linear (r = 0.9995) for standard rutin over the concentration range of 5.0-15.0 microg/mL with specificity for total bioflavonoids (expressed as rutin) at 361.0 nm with an absence of interferents from the complex matrix; RSD of < or = 1.79%, intrarun (E = 97.88 +/- 1.75 to 99.0 +/- 0.33%) and interrun (E = 98.38 +/- 1.12 to 100.79 +/- 1.30%) accuracy; Rec. = 98.64 +/- 0.42 to 100.74 +/- 0.41%; LOD = 0.20 microg/mL; and LOQ = 0.30 microg/mL.     

A comparative study of first-derivative spectrophotometry and column high-performance liquid chromatography applied to the determination of repaglinide in tablets and for dissolution testing

A fast and reliable method for the determination of repaglinide is highly desirable to support formulation screening and quality control. A first-derivative UV spectroscopic method was developed for the determination of repaglinide in tablet dosage form and for dissolution testing. First-derivative UV absorbance was measured at 253 nm. The developed method was validated for linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ) in comparison to the U.S. Pharmacopeia (USP) column high-performance liquid chromatographic (HPLC) method. The first-derivative UV spectrophotometric method showed excellent linearity [correlation coefficient (r) = 0.9999] in the concentration range of 1-35 microg/mL and precision (relative standard deviation < 1.5%). The LOD and LOQ were 0.23 and 0.72 microg/mL, respectively, and good recoveries were achieved (98-101.8%). Statistical comparison of results of the first-derivative UV spectrophotometric and the USP HPLC methods using the t-test showed that there was no significant difference between the 2 methods. Additionally, the method was successfully used for the dissolution test of repaglinide and was found to be reliable, simple, fast, and inexpensive.     

Liquid chromatographic determination of alpha-solasonine in frozen green peas as an indicator of the presence of nightshade berries

Nightshade berries containing glycoalkaloids can be a contaminant in green peas. Methodology was developed to detect this contamination. The glycoalkaloid alpha-solasonine was extracted from frozen green peas with 1% (v/v) acetic acid, cleaned up on a C18 cartridge, and determined by liquid chromatography with UV detection at 200 nm. Method performance characteristics for the determination of alpha-solasonine include linearity from 140 to 1500 ng injected (r = 0.9996-0.9999); recovery ranging from 68 to 79%; limit of quantitation (LOQ) = 4.5 ppm (280 ng standard), and limit of detection = 0.64 ppm (40 ng standard). At the LOQ, the expanded uncertainty at 95% confidence was 0.38 x the reported value. The method was applied to the detection of alpha-solasonine in frozen green peas in a 2-year study of 60 samples of frozen green peas from Ontario, Canada. None of the samples contained alpha-solasonine. No unripe berries of Solanum nigrum were detected visually in the samples.     

Determination of vitamin E acid succinate in biodegradable microspheres by reversed-phase high-performance liquid chromatography

A simple, rapid, and reproducible reversed-phase high-performance liquid chromatographic (HPLC) method is applied to the routine assay of vitamin E acid succinate in biodegradable microspheres. Vitamin E acid-succinate-containing poly-(D,L-lactic-co-glycolic acid) microspheres are prepared by the solvent evaporation method. The starting drug-polymer ratio is 1:10 (w/w) and the total amount of drug and polymer processed is always 440 mg. The content of vitamin E acid succinate in the microspheres is evaluated by HPLC. Chromatography is carried out isocratically at 25 degrees C +/- 0.5 degrees C on an Extrasil ODS-2 column with a mobile phase composed of methanol-water (97:3, v/v) (pH 5.6) at a flow rate of 2 mL/min and UV detection at 284 nm. Parameters such as linearity, limits of quantitation (LOQ) and detection (LOD), precision, accuracy, recovery, specificity, and ruggedness are studied as reported in the International Conference on Harmonization guidelines. The stability of vitamin E acid succinate is also studied with satisfactory results after 48 h at 25 degrees C. The method is selective and linear for drug concentrations in the range 15-210 micro g/mL. The LOQ and LOD are 15 and 3 micro g/mL, respectively. The results for accuracy studies are good. Values for coefficient of variation for intra- and interassay are 2.08% and 2.32%, respectively. The mean percentage of vitamin E acid succinate in the recovery studies is 99.52% +/- 0.81%. The mean loading efficiency for microspheres is 96.53% +/- 1.31%.     

Development and single-laboratory validation of a reversed-phase liquid chromatography-electrospray-tandem mass spectrometry method for identification and determination of acrylamide in foods

A rapid and accurate method using reversed-phase liquid chromatography-tandem mass spectrometry interfaced with electrospray was developed for determination of acrylamide in cooked food samples. A simplified sample treatment procedure using an extraction step with acidified water without cleanup was developed. A C18 column with an aqueous formic acid-methanol mixture as the mobile phase was used under isocratic conditions. The method was validated in-house for robustness, limits of detection (LOD) and quantitation (LOQ), linearity, recovery, and accuracy both on standard and baked-product and potato flour matrixes. Good results in the low ppb level were obtained for LOD (< 15 microg/kg) and LOQ (< 25 microg/kg) of acrylamide in samples. Excellent linearity (r2 = 0.999-1.000) was established over 2 orders of magnitude by performing statistical tests. The absence of both constant and proportional systematic errors demonstrated good method accuracy. Excellent results were obtained for intraday repeatability (RSD < 1.5%) and between-day precision (RSD < 5%). Extraction recoveries from food products were calculated in the 97 +/- 3-99 +/- 2% (n = 6) range with a labeled internal standard (13C3-acrylamide). The applicability of the method to determination of acrylamide in cooked food products was demonstrated.     

Determination of tianeptine in tablets by high-performance liquid chromatography with fluorescence detection

A sensitive and selective high-performance liquid chromatographic method has been developed for the determination of tianeptine (Tia) in tablets. The method is based on derivatization of Tia with 4-chloro-7-nitrobenzofurazan (NBD-CI). A mobile phase consisting of acetonitrile-10 mM orthophosphoric acid (pH 2.5; 77 + 23) was used at a flow rate of 1 mL/min on a C18 column. The Tia-NBD derivative was monitored using a fluorescence detector, with emission set at 520 nm and excitation at 458 nm. Gabapentin was selected as an internal standard. Linear calibration graphs were obtained in the concentration range of 45-300 ng/mL. The lower limit of detection (LOD) was 10 ng/mL at a signal-to-noise ratio of 4. The lower limit of quantitation (LOQ) was 45 ng/mL. The relative standard values for intra- and interday precision were <0.46 and <0.57%, respectively. The recovery of the drug samples ranged between 98.89 and 99.85%. No chromatographic interference from the tablet excipients was found. The proposed method was validated in terms of precision, robustness, recovery, LOD, and LOQ. All the validation parameters were within the acceptance range. The proposed method was applied for the determination of Tia in commercially available tablets. The results were compared with those obtained by an ultraviolet spectrophotometric method using t- and F-tests.     

Kinetic release of triptolide after injection of renal-targeting 14-succinyl triptolide-lysozyme in a rat kidney study by liquid chromatography/mass spectrometry

Triptolide (TP) is one of the most important biologically active components of the Chinese herb Tripterygium wilfordii Hook f (TWHf). A novel high-performance liquid chromatography/mass spectrometry (LC/MS) method was developed to study the kinetic release of TP after intravenous injection of the newly synthesized 14-succinyl triptolide-lysozyme (TPS-LZM). The method was validated in terms of selectivity, linearity, precision, accuracy, stability, limit of detection (LOD) and limit of quantification (LOQ). The calibration curve was linear over the concentration range 0.5-400.0 ng/g. The mean recovery of triptolide from spiked samples, in a concentration range of 0.5-400.0 ng/g, was 91.84% (RSD = 3.69%, n = 3). The intra- and inter-assay coefficients of variation were less than 6.38%. The method with simple sample pretreatment and being highly specific and precise, can be used for analysis of triptolide release in rat kidney after intravenous injection of renal-targeting TPS-LZM conjugate. The results showed that, as compared with free TP, TPS-LZM could significantly increase the concentration and prolong the action time of TP in the kidney.     

Rapid method for determination of chlormequat residues in tomato products by ion-exchange liquid chromatography/electrospray tandem mass spectrometry

A rapid method has been devised for the direct determination of chlormequat in tomato samples. No clean-up is required, and analysis uses ion-exchange liquid chromatography/tandem mass spectrometry interfaced with electrospray ionization (LC/ESI-MS/MS). A cation-exchange column was used with an aqueous ammonium acetate/acetonitrile mixture as the mobile phase under isocratic conditions. The method was validated in terms of detection limits (LOD), quantitation limits (LOQ), linearity, recovery, precision and accuracy. Good results in the low micro g kg(-1) level were obtained for the LOD and LOQ of chlormequat in tomato samples. Comparison of solvent and matrix-matched calibration curves demonstrated the absence of significant matrix effects and the feasibility of using external calibration. Linearity was established over two orders of magnitude by performing homoscedasticity and Mandel fitting statistical tests. The absence of both constant and proportional systematic errors was verified by evaluating the recovery function, demonstrating good method accuracy. Excellent precision in terms of intra-day repeatability was calculated (RSD% <3.4). Extraction recoveries from tomato products were calculated, by using a labelled internal standard (d(4)-chlormequat), to be in the 93 +/- 5-99 +/- 7% range. The applicability of the method to the determination of chlormequat residues in tomato products was demonstrated.     

Simultaneous estimation of genistein and daidzein in Pueraria tuberosa (Willd.) DC by validated high-performance thin-layer chromatography (HPTLC) densitometry method

The present study was performed to estimate the concentration of genistein and daidzein in ethanol extract of tubers of Pueraria tuberosa (Indian kudzu or Vidarikanda) and its various fractions (n-hexane, ethyl acetate, n-butanol, and aqueous) by high-performance thin-layer chromatography (HPTLC). The separation of bioactive compounds was performed using mobile phase, toluene:ethyl acetate:acetone:formic acid (20.0:4.0:2.0:1.0) and detected at wavelength 269?nm. The method was validated for linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), etc. by International Conference on Harmonization guidelines. The calibration range was found to be 100–600?ng/band for both the bioactive compounds. Daidzein was separated with an R_f value of 0.39?±?0.02 and genistein with an R_f value of 0.54?±?0.02. Average recovery was 99.96 and 99.90% for genistein and daidzein, respectively. The LOD and LOQ were 14.786 and 44.805?ng, respectively, for genistein, and 9.607 and 29.114?ng, respectively, for daidzein. Both the phytoconstituents were found in ethanol extract and its ethyl acetate fraction only. The developed HPTLC method was simple, precise, robust, specific, rapid, and cost effective and could be used for quality control analysis and quantification of genistein and daidzein in different herbal formulations containing the plant species.     

Quality control of modified xiaoyao san through the determination of 22 active components by ultra-performance liquid chromatography

A simple, sensitive, and reliable ultra-performance liquid chromatography (UPLC) method has been developed for simultaneous determination of 22 major constituents in modified xiaoyao san (MXS), a multiherbal formula. The chromatographic separation was performed on an ACQUITY UPLC BEH C18 column (150 x 2.1 mm, 1.7 microm, particle size), with an aqueous 0.5% acetic acid and acetonitrile mobile phase gradient. The method was validated for linearity (r2 >0.9937), intraday and interday precision (RSD <8.51%), recovery (91.18-107.73%), LOD (0.02-4.17 ng/mL), and LOQ (0.05-12.50 ng/mL). The established method was successfully applied to quantify the 22 marker compounds in MXS, which provided a useful basis of overall evaluation of the quality of MXS.