Quantitative determination of clozapine in serum by instrumental planar chromatography

An instrumental planar chromatographic (HPTLC) method for quantitative analysis of clozapine in human serum was developed and validated. Clozapine was extracted with n-hexane-isoamyl alcohol (75:25 v/v). The chromatographic separation was achieved on precoated silica gel F 254 HPTLC plates using a mixture of chloroform and methanol (9:1 v/v) as mobile phase. Quantitative analyses were carried out by densitometry at a wavelength of 290 nm. The method was linear between 10 and 100 ng/spot, corresponding to 0.10 and 1.00 ng/microL of clozapine in human serum after extraction process and applying 10 microL to the chromatographic plates. The method correlation coefficient was 0.999. The intra-assay variation was between 2.10 and 3.33% (n = 5) and the interassay was between 2.67 and 4.44% (n = 9). The detection limit was 0.03 ng/microL, and the quantification limit was 0.05 ng/microL. The method proved to be accurate, with a recovery between 97.00 and 99.00%, with an RSD not higher than 7.22%, and was selective for the active principle tested. This method was successfully applied to quantify clozapine in patient serum samples. In conclusion, the method is useful for the quantitative determination of clozapine in serum.     

Quantitative determination of haloperidol in tablets by high performance thin-layer chromatography

A densitometric high performance thin-layer chromatography (HPTLC) method was developed and validated for the quantitative analysis of haloperidol in tablets. Chromatographic separation was achieved on precoated silica gel F 254 HPTLC plates using a mixture of acetone/chloroform/n-butanol/acetic acid glacial/water (5:10:10:2.5:2.5 v/v/v/v/v) as the mobile phase. Quantitative analysis was carried out at a wavelength of 254 nm. The method was linear in the 10-100 ng/microL range, with a determination coefficient of 0.999. The coefficients of variation for precision were not higher than 2.35%. The detection limit was 0.89 ng/microL, and the quantification limit was 2.71 ng/microL. The accuracy ranged from 97.76 to 100.33%, with a CV not higher than 4.50%. This method was successfully applied to quantify haloperidol in real pharmaceutical samples, including the comparison with HPLC measurements. The method was fast, specific, with a good precision and accuracy for the quantitative determination of haloperidol in tablets.     

Comparison of high performance TLC and HPLC for separation and quantification of chlorogenic acid in green coffee bean extracts

Two chromatographic methods, high-performance TLC (HPTLC) and HPLC, were developed and used for separation and quantitative determination of chlorogenic acid in green coffee bean extracts. For HPTLC silica gel Kieselgel 60 F 254 plates with ethyl acetate/dichlormethane/formic acid/acetic acid/water (100:25:10:10:11, v/v/v/v/v) as mobile phase were used. Densitometric determination of chlorogenic acid by HPTLC was performed at 330 nm. A gradient RP HPLC method was carried out at 330 nm. All necessary validation tests for both methods were developed for their comparison. There were no statistically significant differences between HPLC and HPTLC for quantitative determination of chlorogenic acid according to the test of equality of the means.     

Ten marker compounds-based comparative study of green tea and guava leaf by HPTLC densitometry methods: antioxidant activity profiling

High-performance thin layer chromatography (HPTLC) method for the separation and quantitative determination of ten markers (catechins, flavonoids, and phenolics) in different extracts of green tea and guava leaf has been developed and the antioxidant activity profiles of the two plant extracts have been determined. Ten marker compounds have been resolved using silica gel 60 F(254) plates, toluene/acetone/formic acid (5:4:1 v/v/v) for markers 1-6, and toluene/ethyl acetate/formic acid/methanol (3:3:0.8:0.2 v/v/v/v) for markers 7-10 as the mobile phases. The high-performance thin layer chromatography densitometry was performed at wavelengths of 282 and 285 nm for the markers 1-6 and 7-10, respectively. Potent antioxidant activity and the presence of phenolics and flavan-3-ols has been observed for the guava leaf extracts suggestive of its use as an alternate economical source of antioxidants over green tea--the well-established food additive/nutraceutical agent.     

Estimation of berberine in ayurvedic formulations containing Berberis aristata

A sensitive, simple, rapid, and efficient high-performance thin-layer chromatographic (HPTLC) method has been developed and validated for the analysis of berberine in marketed Ayurvedic formulations containing Berberis aristata DC for regulatory purposes. Chromatography of methanolic extracts of these formulations was performed on silica gel 60 F254 aluminum-backed TLC plates of 0.2 mm layer thickness. The plate was developed up to 66 mm with the ternary-mobile phase butanol-acetic acid-water (8 + 1 + 1, v/v/v) at 33 +/- 5 degrees C with 5 min of tank saturation. The marker, berberine, was quantified at its maximum absorbance of 350 nm. The limit of detection and limit of quantitation values were found to be 5 and 10 ng/spot. The linear regression analysis data for the calibration plot showed a good linear relationship with correlation coefficient = 0.9994 in the concentration range of 10 to 50 ng/spot for berberine with respect to peak area. The instrumental precision was found to be 0.49% coefficient of variation (CV), and repeatability of the method was 0.73% CV. Recovery values from 98.27 to 99.11% indicate excellent accuracy of the method. The developed HPTLC method is very accurate, precise, and cost-effective, and it has been successfully applied to the assay of marketed formulations containing B. aristata for determination of berberine.     

Simultaneous determination of atorvastatin calcium and ramipril in capsule dosage forms by high-performance liquid chromatography and high-performance thin layer chromatography

Two simple and accurate methods to determine atorvastatin calcium and ramipril in capsule dosage forms were developed and validated using HPLC and HPTLC. The HPLC separation was achieved on a Phenomenex Luna C18 column (250 x 4.6 mm id, 5 microm) in the isocratic mode using 0.1% phosphoric acid-acetonitrile (38 + 62, v/v), pH 3.5 +/- 0.05, mobile phase at a flow rate of 1 ml/min. The retention times were 6.42 and 2.86 min for atorvastatin calcium and ramipril, respectively. Quantification was achieved with a photodiode array detector set at 210 nm over the concentration range of 0.5-5 microg/mL for each, with mean recoveries (at three concentration levels) of 100.06 +/- 0.49% and 99.95 +/- 0.63% RSD for atorvastatin calcium and ramipril, respectively. The HPTLC separation was achieved on silica gel 60 F254 HPTLC plates using methanol-benzene-glacial acetic acid (19.6 + 80.0 + 0.4, v/v/v) as the mobile phase. The Rf values were 0.40 and 0.20 for atorvastatin calcium and ramipril, respectively. Quantification was achieved with UV densitometry at 210 nm over the concentration range of 50-500 ng/spot for each, with mean recoveries (at three concentration levels) of 99.98 +/- 0.75% and 99.87 +/- 0.83% RSD for atorvastatin calcium and ramipril, respectively. Both methods were validated according to International Conference on Harmonization guidelines and found to be simple, specific, accurate, precise, and robust. The mean assay percentages for atorvastatin calcium and ramipril were 99.90 and 99.55% for HPLC and 99.91 and 99.47% for HPTLC, respectively. The methods were successfully applied for the determination of atorvastatin calcium and ramipril in capsule dosage forms without any interference from common excipients.     

Estimation of L-dopa from Mucuna pruriens LINN and formulations containing M. pruriens by HPTLC method

A selective, precise, and accurate high-performance thin-layer chromatographic (HPTLC) method has been developed for the analysis of L-dopa in Mucuna pruriens seed extract and its formulations. The method involves densitometric evaluation of L-dopa after resolving it by HPTLC on silica gel plates with n-butanol-acetic acid-water (4.0+1.0+1.0, v/v) as the mobile phase. Densitometric analysis of L-dopa was carried out in the absorbance mode at 280 nm. The relationship between the concentration of L-dopa and corresponding peak areas was found to be linear in the range of 100 to 1200 ng/spot. The method was validated for precision (inter and intraday), repeatability, and accuracy. Mean recovery was 100.30%. The relative standard deviation (RSD) values of the precision were found to be in the range 0.64-1.52%. In conclusion, the proposed TLC method was found to be precise, specific and accurate and can be used for identification and quantitative determination of L-dopa in herbal extract and its formulations.     

Determination of cimetidine,famotidine, and ranitidine hydrochloride in the presence of their sulfoxide derivatives in pure and dosage forms by high-performance thin-layer chromatography and scanning densitometry

A selective, precise, and accurate method was developed for the determination of cimetidine (C), famotidine (F), and ranitidine hydrochloride (R x HCl) in the presence of their sulfoxide derivatives. The method involves quantitative densitometric evaluation of mixtures of the drugs and their derivatives after separation by high-performance thin-layer chromatography on silica gel plates (10 x 20 cm) with ethyl acetate-isopropanol-20% ammonia (9 + 5 + 4, v/v) as the mobile phase for both C and F and ethyl acetate-methanol-20% ammonia (10 + 2 + 2, v/v) as the mobile phase for R x HCl; Rf values for C, F, and R x HCl and their corresponding derivatives were 0.85 and 0.59, 0.73 and 0.41, and 0.56 and 0.33, respectively. Developing time was approximately 20 min. For densitometric evaluation, peak areas were recorded at 218, 265, and 313 nm for C, F, and R x HCl, respectively. The relationship between concentration and the corresponding peak area was plotted for the ranges of 5-50 microg/spot for C and 2-20 microg/spot for F and R x HCl. Mean recoveries were 100.39 +/- 1.33, 99.77 +/- 1.30, and 100.09 +/- 0.69% for C, F, and R x HCl, respectively. The proposed method was used successfully for stability testing of the pure drugs in the presence of up to 90% of their degradates, in bulk powder and dosage forms. The results obtained were analyzed statistically and compared with those obtained by the official methods.     

柱前衍生-超高效液相色谱-串联四极杆质谱法测定茶叶中乙撑硫脲的残留

建立了茶叶中乙撑硫脲残留的柱前衍生-超高效液相色谱-串联四极杆质谱检测方法。样品采用乙腈提取,提取液经QuEChERS基质分散固相萃取净化后采用9-芴基甲基氯甲酸酯（FMOC-CL）柱前衍生;衍生溶液经BEH-C18色谱柱（100 mm×2.0 mm,1.7 μm）分离后进入串联四极杆质谱仪检测,采用同位素内标法定量;流动相为0.1%（v/v）甲酸-乙腈。该方法对茶叶样品检出限为1.3 μg/kg,定量限为4.2 μg/kg;加标回收率在97.7%~107.5%之间,相对标准偏差（RSD,n=6）在2.1%~10.0%之间;在1.0~203.4 μg/L范围内线性回归系数r为0.9993。该方法灵敏度高,重现性好,定性定量准确,可有效满足对茶叶中乙撑硫脲残留检测的要求。     

A validated HPTLC method for determination of trans-caryophyllene from polyherbal formulations

Formulations of traditional medicines are usually made up of a complex mixture of herbs. However, effective quality control methods in order to select materials of the right quality are lacking. 'Amukkara choornam' is a polyherbal Siddha formulation used for gastritis, spleen enlargement, leucorrhoea, hiccups, anaemia, tuberculosis and kappa diseases. Trans-caryophyllene is an important constituent present in the ingredients of this formulation. In a literature survey, it was found that there is no such method for the quantification of trans-caryophyllene except gas chromatography or gas chromatography-mass spectroscopy (GC-MS). So, a high performance thin layer chromatography (HPTLC) method was developed and validated for the quantification of trans-caryophyllene in amukkara choornam. Pre-coated silica gel 60F-254 plates (10 × 10 cm2) were used for the analysis. The solvent system consisted of toluene-ethyl acetatate (9 : 3, v/v), and trans-caryophyllene was detected at 260 nm. The developed method was validated for linearity (R2 = 0.9996 ± 0.0034), limit of detection (LOD) (0.101 ng), limit of quantification (LOQ) (0.639 ng), accuracy (% recovery = 97.19 ± 1.204), and precision (CV < 5%, for both intra-day and inter-day precisions). The levels of trans-caryophyllene were found to be 3.5-4.10 μg per gram of herbal products.     

Assay of tinidazole in human serum by high-performance thin-layer chromatography--comparison with high-performance liquid chromatography

Determination of tinidazole in human serum by high-performance thin-layer chromatography (HPTLC) is presented. It includes use of 10 x 10 cm plates coated with silica gel 60 and chloroform-acetonitrile-acetic acid (60 + 40 + 2) as mobile phase. Quantitation was performed by densitometry at 320 nm. The linearity (1-10 ng), precision (6%), reproducibility (5%), recovery (96%), and detection limit (1 mg/L) of tinidazole determination by HPTLC were comparable with corresponding method parameters by reversed-phase HPLC. A satisfactory correlation was found between the 2 analytical methods. The procedure was used to quantitate tinidazole in patient sera.     

Chemical stability of haloperidol injection by high performance thin-layer chromatography

The chemical stability of haloperidol lactate injection was studied under different storage conditions by high performance thin-layer chromatography (HPTLC). The study was performed at 25 +/- 2 degrees C and at refrigeration temperature (8 +/- 1 degrees C) in original glass ampoules over 15 days after being opened. The samples tested at 25 +/- 2 degrees C were stored with exposure to and protection from light. Chromatographic separation was achieved on precoated silica gel F 254 HPTLC plates using a mixture of acetone/chloroform/n-butanol/glacial acetic acid/water (5:10:10:2.5:2.5, v/v/v/v/v) as a mobile phase. Quantitative analyses were carried out at a wavelength of 254 nm. The method exhibited adequate linearity (r = 0.999), selectivity, precision (RSD = 1.92%), and accuracy (recoveries from 98.59 to 101.90%). The concentrations of all samples remained greater than or 90% of the original concentration. Haloperidol lactate injection was chemically stable under all conditions studied over 15 days.     

Validation of a reversed phase high performance thin layer chromatographic-densitometric method for secoisolariciresinol diglucoside determination in flaxseed

The validation of a HPTLC-densitometric method for the determination of secoisolariciresinol diglucoside (SDG) in flaxseed was performed improving the reproducibility of a previously reported HPTLC densitometric procedure by the use of fully wettable reversed phase plates (silica gel 60 RP18W F(254S), 10cmx10cm) with MeOH:HCOOH 0.1% (40:60, v/v) mobile phase. The analysis required only the alkaline hydrolysis in aqueous medium of undefatted samples and densitometry at 282nm of HPTLC runs. The method was validated following the protocol proposed by the Société Francaise des Sciences et Techniques Pharmaceutiques (SFSTP) giving rise to a dependable and high throughput procedure well suited to routine application. SDG was quantified in the range of 321-1071ng with RSD of repeatability and intermediate precision not exceeding 3.61% and accuracy inside the acceptance limits. Flaxseed of five cultivars of different origin was elected as test-bed.     

High-performance thin layer chromatography method for quantitative determination of four major anthraquinone derivatives in Rheum emodi

A high-performance thin layer chromatographic (HPTLC) method for the rapid and simple quantification of the four major anthraquinone derivatives i.e. physcion, chrysophanol, emodin and chrysophanol glycoside in Rheum emodi is described. HPTLC of anthraquinone derivatives was performed on pre-coated RP-18 F254S HPTLC plates. For achieving good separation, the mobile phase of methanol-water-formic acid (80:19:1, v/v/v) was used. The densitometric determination of anthraquinone derivatives was carried out at 445 nm in reflection/absorption mode. The calibration curves were linear in the range of 20-100 ng for physcion, 80-400 ng for chrysophanol and emodin, and 200-1000 ng for chrysophanol glycoside. The method was found to be reproducible and convenient for quantitative analysis of anthraquinone derivatives in the methanolic extract of rhizomes of R. emodi collected from three different locations of Western Himalaya, India.     

Quality assessment for tramadol in pharmaceutical preparations with thin layer chromatography and densitometry

Research studies have been carried out to develop a chromatographic and densitometric method suitable for identification and determination of tramadol and impurities. In addition, the stability of tramadol in solutions was investigated, including an effect of solution pH, temperature and incubation time. In the first instance the conditions for identification and quantitative determination of tramadol and impurities in pharmaceutical preparations were established. The separation was performed on silica gel-coated chromatographic plates (HPTLC) using two mobile phases: (I) chloroform-methanol-glacial acetic acid (9:2:0.1, v/v/v); (II) chloroform-toluene-ethanol (9:8:1, v/v/v). The UV densitometry was carried out at lambda = 270 nm. The developed method is of high sensitivity and low detection and determination limits ranging from 0.044 to 0.35 microg. For individual constituents the recovery ranges from 93.23 to 99.66%. The next step was to evaluate the stability of tramadol and determine a method of decomposition under various experimental conditions. It was found that tramadol decomposes in various ways in acidic and basic environments producing (1RS)-[2-(3-methoxyphenyl)cyclohex-2-enyl]-N,N-dimethylmethanamine (imp. B) and (1RS, 2RS)-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl)cyclohexanol (imp. cis-T) or imp. cis-T, respectively.     

Thin Layer Chromatographic Method for Rapid Quantification and Identification of Trimethoprim and Sulfamethoxazole in Pharmaceutical Dosage Forms

Abstract

A densitometric and a spectrophotometric method for rapid but accurate determination of sulfamethoxazole (SMA) and trimethoprim (TMP) present in combined dosage forms were described. SMA and TMP were extracted with 90% acqueous methanol and the interfering and related contaminants were removed by thin layer chromatography (TLC) or high performance TLC (HPTLC) on silicagel plates using chloroform : isopropanol : diethylamine :: 10 : 6 : 1 (v/v) as mobile phase. Assay was done at the respective absorption maxima of the drugs by in situ densitometry and by spectroscopy after extracting the drugs from TLC plates with 90% acqueous ethanol. Results obtained by both the methods agreed well with those obtained by the method prescribed by the United States Pharmacopoeia XXI edition. Total time required for HPTLC and densitometric assay of 32 samples using 4 standards was 30 min. Probable source of errors in densitometric studies and their rectification was discussed.     

Determination of Pesticide Residues in Teas via QuEChERS Combined with Dispersive Liquid–Liquid Microextraction Followed by Gas Chromatography–Tandem Mass Spectrometry

In this study, an effective gas chromatography–tandem mass spectrometry method was developed to determine 47 pesticide residues in tea. Sample preparation involved a quick, easy, cheap, effective, rugged, and safe (QuEChERS) procedure, wherein the sample is extracted by acetonitrile and cleaned up with multiwalled carbon nanotubes and primary secondary amine adsorbents; dispersive liquid–liquid microextraction (DLLME) was subsequently performed using carbon tetrachloride as extractive solvent and the extract obtained by QuEChERS as dispersive solvent. Factors influencing DLLME efficiency, including type and volume of extractive solvent, volume of dispersive solvent, and extraction time were evaluated. For validation purposes, recovery studies were performed using matrix blanks fortified with pesticides at three concentrations, namely, 10, 50, and 100 μg kg^?1. Most of the analytes were recovered at an acceptable range of 70?120% and RSDs ≤ 20% were acquired for green tea, oolong tea, black tea, and puer tea. Limits of quantification of pesticides obtained for these teas were sufficiently low, and most pesticides levels were lower than 10 μg kg^?1, which satisfies the requirements for maximum residue levels (MRLs) as prescribed by the European Community. Twenty-four commercially available tea samples were analyzed using this optimized method. Results revealed that the contents of chlorpyrifos and alpha-HCH from different green tea samples exceed the MRLs, and chlorpyrifos, bifenthrin, lambda-cyhalothrin, and cypermethrin are among the most frequently detected pesticides in teas.     

Quantitative determination of canagliflozin in human plasma samples using a validated HPTLC method and its application to a pharmacokinetic study in rats

Canagliflozin (CNZ) is the first sodium–glucose co-transporter-2 inhibitor approved for treatment of type 2 diabetes mellitus. In the proposed work, a sensitive, rapid and validated high-performance thin-layer chromatography (HPTLC) method was established for the estimation of CNZ in human plasma for the first time. HPTLC analysis of CNZ and internal standard (sildenafil) was performed on glass coated silica gel 60 F₂₅₄ HPTLC plates using a binary mixture of chloroform–methanol 9:1 (%, v/v) as the mobile phase. Densitometric detection was done at 295 nm. Retardation factor values were obtained as 0.22 and 0.52 for the CNZ and the IS, respectively. The linearity range of CNZ was obtained as 200–3,200 ng/ml. A simple protein precipitation method was used for the extraction of analyte from plasma using methanol. The proposed HPTLC technique was validated for linearity, accuracy, precision and robustness. The proposed HPTLC technique was successfully utilized for the assessment of pharmacokinetic profile of CNZ in rats after oral administration. After oral administration, the peak plasma concentration of CNZ was obtained as 1458.01 ng/ml in 2 h. The proposed HPTLC method could be applied to the study of the pharmacokinetic profile of pharmaceutical formulations containing CNZ.     

基质分散固相萃取-超高效液相色谱-串联质谱法测定蔬菜风险监测中的9种农药残留

建立了基质分散固相萃取-超高效液相色谱-串联质谱测定蔬菜中多菌灵、氧乐果、克百威、涕灭威、毒死蜱、甲胺磷、甲拌磷、对硫磷、甲基对硫磷9种农药残留的方法。蔬菜通过乙腈提取、盐析分配、基质分散固相萃取净化后,采用Waters BEH C18柱(100 mm×2.1 mm, 1.7 μm),以乙腈和0.1%甲酸水溶液作为流动相,梯度洗脱,电喷雾电离正离子(ESI⁺)、多反应监测(MRM)模式测定,基质匹配标准溶液工作曲线法定量。该方法的检出限为0.8~4.0 μg/kg,回收率为72.8%~117.4%。50批蔬菜样品中毒死蜱、多菌灵和氧乐果残留的检出率分别为42.0%、14.0%和2.0%,毒死蜱超标率为8.0%,其他农药未检出。该法可同时测定食品风险监测中蔬菜的农药残留,具有操作方便、准确率高、重复性好等优点,可满足蔬菜中农药残留的检测要求。     

Stability-indicating high-performance thin-layer chromatographic determination of levonorgestrel and ethinyloestradiol in bulk drug and in low-dosage oral contraceptives

A stability-indicating high-performance thin-layer chromatography (HPTLC) method was developed and validated for simultaneous determination of steroidal hormones levonorgestrel and ethinyloestradiol both in bulk drug and in low-dosage oral contraceptives. Optimization of conditions for the spectrodensitometric procedure was reached by eluting HPTLC silica gel plates in a 10 cm × 10 cm horizontal chamber. The solvent system consisted of hexane-chloroform-methanol (1.0:3.0:0.25, v/v/v). This system was found to give compact, dense and typical peaks for both levonorgestrel (R_f = 0.65 ± 0.03) and ethinyloestradiol (R_f = 0.43 ± 0.02). Densitometric analysis of the drugs was carried out in the reflectance mode at 225 nm by using a computer controlled densitometric scanner. The calibration curves of levonorgestrel and ethinyloestradiol were linear in the range of 200-800 and 40-160 ng per spot, respectively. The method was validated for precision, robustness and recovery. As the proposed method can effectively separate the drugs from their degradation products, it can be employed as a stability-indicating method.