Development and validation of a high-performance thin-layer chromatographic method for determination of ofloxacin residues on pharmaceutical equipment surfaces

An HPTLC method with densitometric quantification using fluorescence at 313 nm was developed and validated for the determination of ofloxacin residue in controlling pharmaceutical equipment cleanliness. Simulated samples at a residue level of 1 mg/m2 were prepared by spreading the calculated amount of ofloxacin solution on 1, 5, and 10 dm2 stainless steel surfaces. After evaporation of the solvent, the residue was removed by two ethanol wetted cotton swabs, which were thereafter extracted with the mixture of ethanol and Na2EDTA-water solution at pH 11 for 15 min with sonication. The extract and standards were applied on HPTLC silica gel 60 plates and then developed in a horizontal developing chamber from both sides using ethanol-conc. ammonia (4+1, v/v) as the mobile phase. The mean recovery (n=6) at 1 mg/m2 from 1, 5, and 10 dm2 was 95.3, 88.6, and 89.7% with the CV values 3.78, 4.41, and 4.97%, respectively. The absolute detection limit was 0.6 ng and the quantitation limit was 2 ng, but it was shown that these can be improved by immersion of the developed plate into a solution of liquid paraffin-n-hexane (1+2, v/v) to approximately 0.25 and 0.9 ng, respectively. The LOD of the method using detection without paraffin-n-hexane was 3, 0.6, and 0.3 microg/m2 by swabbing 1, 5, and 10 dm2, respectively. The method can be applied to routine control of pharmaceutical equipment cleanliness by sampling from stainless steel surface areas of 1 to 10 dm2 with acceptable residue limit/surface of 1 mg/m2.     

TLC determination of mebendazol and pentoxifylline as residues on pharmaceutical equipment surfaces

This paper presents the applicability of thin-layer chromatographic methods with a subsequent densitometric or video densitometric quantitation for determination of residues in controlling pharmaceutical equipment cleanliness. Analytical methods were developed for monitoring residues of pentoxifylline at 10 mg/M2 and mebendazol at 1 mg/M2 on stainless steel surfaces. Simulated samples were prepared by addition of a calculated amount of pharmaceutical (as a solution) on a 35 x 35 cm stainless steel surface. After evaporation of solvent, the residues were wiped with wetted cotton. The cotton was extracted with dichloromethan-methanol (1 + 1). Filtered extract was concentrated by vacuum evaporation and an aliquot applied to the plate, where standards were also applied. In the narrow concentration range near the acceptable residue limits, linear calibration curve could be obtained for both substances. The mean recovery (n = 4) obtained by densitometric quantitation was 93.4% for pentoxifylline and 85.6% for mebendazol, with coefficients of variation of 3.5 and 8.3%, respectively. Results of video densitometric quantitation did not differ significantly. However, data acquisition and evaluation is faster compared with densitometry and allows better archiving possibilities as required by the regulatory authorities. Both quantitation modes can be applied to routine control of pharmaceutical equipment cleanliness.     

Rapid,simple, and highly sensitive analysis of drugs in biological samples using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry

Rapid and precise identification of toxic substances is necessary for urgent diagnosis and treatment of poisoning cases and for establishing the cause of death in postmortem examinations. However, identification of compounds in biological samples using gas chromatography and liquid chromatography coupled with mass spectrometry entails time-consuming and labor-intensive sample preparations. In this study, we examined a simple preparation and highly sensitive analysis of drugs in biological samples such as urine, plasma, and organs using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry (TLC/MALDI/MS). When the urine containing 3,4-methylenedioxymethamphetamine (MDMA) without sample dilution was spotted on a thin-layer chromatography (TLC) plate and was analyzed by TLC/MALDI/MS, the detection limit of the MDMA spot was 0.05 ng/spot. The value was the same as that in aqueous solution spotted on a stainless steel plate. All the 11 psychotropic compounds tested (MDMA, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, methamphetamine, p-hydroxymethamphetamine, amphetamine, ketamine, caffeine, chlorpromazine, triazolam, and morphine) on a TLC plate were detected at levels of 0.05 − 5 ng, and the type (layer thickness and fluorescence) of TLC plate did not affect detection sensitivity. In addition, when rat liver homogenate obtained after MDMA administration (10 mg/kg) was spotted on a TLC plate, MDMA and its main metabolites were identified using TLC/MALDI/MS, and the spots on a TLC plate were visualized by MALDI/imaging MS. The total analytical time from spotting of intact biological samples to the output of analytical results was within 30 min. TLC/MALDI/MS enabled rapid, simple, and highly sensitive analysis of drugs from intact biological samples and crude extracts. Accordingly, this method could be applied to rapid drug screening and precise identification of toxic substances in poisoning cases and postmortem examinations.     

Simple method of paroxetine determination using a single channel FIA with no in-line reaction process.

A flow-injection analysis (FIA) of paroxetine hydrochloride (PRX), a selective serotonin reuptake inhibitor (SSRI) currently used as an antidepressant drug, is described. A 0.1 mol dm(-3) acetate buffer at pH 3.07 was found to be thebest solvent. The analyte was detected at 293 nm. The calibration equation was linear over the range of 1.07 x 10(-6) to 5.35 x 10(-6) mol dm(-3). The limit of detection (LOD) and the limit of quantitation (LOQ) were 3.2 x 10(-7) and 9.5 x 10(-7) mol dm(-3), respectively. The proposed method was applied to the determination of PRX in pharmaceutical preparations. The results were compared with those obtained by a conventional batchwise UV-spectrophotometry.     

药物分析中薄层色谱的方法认证

在药物分析中,针对所要求的性能参数,对一个薄层色谱程序的各个环节必须进行的认证方法和认可标准进行了讨论。建议当提出结果报告时,应附上关于对方法的认证参数和认证方法的说明。     

Determination of chloramphenicol residue in fish and shrimp tissues by gas chromatography with a microcell electron capture detector

A gas chromatography method with microcell electron capture detection was developed for the determination of chloramphenicol residue in fish and shrimp muscle tissues. The tissue samples were extracted with ethyl acetate, defatted with hexane, and derivatized with Sylon BFT [N,O-bis (trimethylsilyl) trifluoroacetamide-trimethylchlorosilane (99 + 1)]. The limit of detection was 0.04 ng/g and the limit of quantitation 0.1 ng/g. Average recoveries were 70.8-90.8% for fish and 69.9-86.3% for shrimp, respectively. The method was validated for the determination of practical samples.     

Quantitative analysis by thin-layer chromatography with secondary ion mass spectrometry

Summary A direct combination of thin-layer chromatography with secondary ion mass spectrometry (TLC/SIMS) provides a method for the quantitative analysis of thermally unstable compounds or compounds of low volatility such as nicergoline. The method is very simple and has excellent precision. The analysis was performed by using an aluminium TLC plate and a mixture of methylene chloride, acetone, and distilled water as a developing solvent. After development the portion of the plate with the nicergoline and the internal standard spots was cut off the TLC plate, and was attached to the SIMS holder directly. The amount of nicergoline was determined from the ratio of the fragment ion intensity of the nicergoline to the internal standard. The calibration curve was linear, and the detection limit was 10 ng at a signal-to-noise ratio of 5. This method should be considered for application to the determination of drugs in biological samples and also for the determination of possible impurities and decomposition products in drugs.     

Effective separation and simultaneous analysis of anabolic androgenic steroids (AAS) in their pharmaceutical formulations by a validated TLC-densitometry method

ABSTRACT: A TLC densitometric method was developed for simultaneous determination of four anabolic androgenic steroids (AAS) of testosterone derivatives including testosterone propionate (TP), testosterone phenyl propionate (TPP), testosterone isocaproate (TI) and testosterone deaconate (TD) in their pharmaceutical products. Separation was carried out on Al based TLC plates, pre-coated with silica gel 60F-254 using hexane and ethyl acetate (8.5:1.5, v/v). Spots at Rf 0.31+/-0.01, 0.34+/-0.01, 0.40+/-0.01 and 0.45+/-0.02 were recognized as TPP, TP, TI and TD, respectively. Quantitative analysis was done by densitometric measurements at lambdamax 251 nm for all derivatives. The developed method was validated as per ICH guidelines. Method was found linear over the concentration range of 200-1200 ng/spot with the correlation coefficient of 0.995, 0.993, 0.995 and 0.996 for TP, TPP, TI, TD, respectively. Limit of detection for all derivatives were in the range of 16.7-22.3 ng/spot while limit of quantitation were found to be in the range of 55.7-70.9 ng/spot. The developed TLC method can be applied for the simultaneous routine analysis of testosterone derivatives in their individual and combined pharmaceutical formulations.     

分散液-液微萃取结合离子迁移谱测定人体唾液中的苯海拉明

建立一种结合便携式高分辨离子迁移谱(IMS)快速简便萃取唾液中的苯海拉明(DPH)的分散液-液微萃取法(DLLME).取2 mL唾液,采用10％(w:V)硫酸锌沉淀蛋白,以2 mL二氯甲烷为萃取剂对唾液中的苯海拉明进行萃取,萃取时间5 min,离心(3500 r/min,20 min),取二氯甲烷层,45℃真空干燥,甲...     

Determination of glibenclamide in human serum by HPLC

The antidiabetic drug glibenclamide can be reliably quantitated in human serum with high performance liquid chromatography. The serum is buffered and extracted with toluene. The organic solvent is evaporated, the residue dissolved in the mobile phase and an aliquot sampled automatically and chromatographed. UV-detection at 229 m allows a lower limit of quantitation of 5 ng/ml. Precise handling of exact volumes facilitates external calibration. Statistical data for imprecision and inaccuracy are given and illustrate reliable quantification. Application of the method to experimental and clinical pharmacokinetic studies with specific problems is illustrated.     

气相色谱-负化学离子源质谱法检测胡萝卜中残留的环氟菌胺

建立了胡萝卜中环氟菌胺残留量的气相色谱-负化学离子源质谱(GC-NCI/MS)检测方法。用乙酸乙酯对胡萝卜中的环氟菌胺进行提取,并经固相萃取(SPE)净化后,由GC-NCI/MS在选择离子监测模式(SIM)下测定。该方法的准确度和精密度较高,在0.005,0.01,0.02,0.04 mg/kg 4个加标水平下,环氟菌胺的平均回收率均处于74.9%～96.4%之间,相对标准偏差(RSD)小于9.7%。在10～1000ng/mL范围内线性关系良好,检出限为0.001 mg/kg,定量限为0.005 mg/kg。该方法选择性好,抗干扰能力强,可作为胡萝卜中环氟菌胺残留检测的确证方法。     

Stress degradation studies and development of stability-indicating TLC-densitometry method for determination of prednisolone acetate and chloramphenicol in their individual and combined pharmaceutical formulations

ABSTRACT: A rapid and reproducible stability indicating TLC method was developed for the determination of prednisolone acetate and chloramphenicol in presence of their degraded products. Uniform degradation conditions were maintained by refluxing sixteen reaction mixtures for two hours at 80°C using parallel synthesizer including acidic, alkaline and neutral hydrolysis, oxidation and wet heating degradation. Oxidation at room temperature, photochemical and dry heating degradation studies were also carried out. Separation was done on TLC glass plates, pre-coated with silica gel 60F-254 using chloroform: methanol (14:1 v/v). Spots at Rf 0.21 ± 0.02 and Rf 0.41 ± 0.03 were recognized as chloramphenicol and prednisolone acetate, respectively. Quantitative analysis was done through densitometric measurements at multiwavelength (243 nm, λmax of prednisolone acetate and 278 nm, λmax of chloramphenicol), simultaneously. The developed method was optimized and validated as per ICH guidelines. Method was found linear over the concentration range of 200-6000 ng/spot with the correlation coefficient (r2 ± S.D.) of 0.9976 ± 3.5 and 0.9920 ± 2.5 for prednisolone acetate and chloramphenicol, respectively. The developed TLC method can be applied for routine analysis of prednisolone acetate and chloramphenicol in presence of their degraded products in their individual and combined pharmaceutical formulations.     

Determination of sodium cromoglycate in human plasma by liquid chromatography with tandem mass

A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of sodium cromoglycate (SCG) in human plasma after a nasal dose of 10.4 mg sodium cromoglycate nasal spray, using pravastatin sodium as the internal standard. The method was validated over a linear range of 0.300-20.0 ng/mL. SCG and I.S. were extracted from 1.0 mL of heparinized plasma by C(18) solid-phase extraction cartridges using methanol as eluting solvent. The dried residue was reconstituted with 100 microL of mobile phase, and 10 microL was injected onto the LC-MS/MS system. Chromatographic separation was achieved on a C(18) column (250 x 4.6 mm i.d., 5 microm particle size) with a mobile phase of methanol-acetonitrile-water (containing 2 mmol/L ammonium acetate; 42.5:42.5:15, v/v/v) at a flow rate of 0.4 mL/min. The analytes were detected with a triple quad LC-MS/MS using ESI with positive ionization. Ions monitored in the multiple reaction monitoring mode were m/z 469.0 (precursor ion) to m/z 245.0 (product ion) for SCG and m/z 447.2 (precursor ion) to m/z327.1 (product ion) for pravastatin sodium (internal standard) The average recovery of SCG from human plasma was 94.88% and the lower limit of quantitation was 0.3 ng/mL. Results from a 3-day validation study demonstrated excellent precision and accuracy across the calibration range of 0.3-20 ng/mL. The method was successfully applied to the pharmacokinetic study of SCG in healthy Chinese volunteers. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Determination of 8-hydroxyquinoline sulfate in tuberculin solutions by planar and high performance liquid chromatography

Summary TLC and HPLC methods for the determination of the preservative, 8-hydroxyquinoline sulfate in PPD-T tuberculin solution were developed. The planar chromatography method involved separation of 8-hydroxyquinoline sulfate on a TLC plate using a butyl-acetate: formic acid: 2-propanol mobile phase, detection and quantitation by densitometric scanning. The HPLC method was on a LiChrosorb RP-18 column with acetonitrile-water (65:35 v/v) mobile phase, adjusted to pH 3.05 by phosphoric acid. Linearity, reproducibility and accuracy were found to be satisfactory. Under selected conditions, the limit of detection (LOD) of both methods was similar-about 25 ng. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Determination of residual clopidol in chicken muscle by capillary gas chromatography/mass spectrometry

Clopidol was extracted from chicken with acetonitrile, and the extract was evaporated to about 5 mL. The concentrated solution was centrifuged and applied to a glass column filled with basic alumina. The column was eluted with methanol and the methanol was evaporated to dryness. The residue was derivatized with Sylon BFT and analyzed by gas chromatography/mass spectrometry (GC/MS). Mass characteristics of trimethylsilyl derivative of clopidol were interpreted manually. The selected-ion monitoring mode was performed at m/z 191, 248, 263, and 265. A sensitive, reproducible GC/MS method was developed for monitoring residues of clopidol in chickens. All recoveries of the drug from chicken muscle fortified at 1, 10, and 100 ng/g were > 65%, and relative standard deviations were < 15%. The detection limit was about 0.5 ng/g, and the quantitation limit was the lower limit of the standard curve, which was about 1 ng/g.     

Sensitive high-performance liquid chromatographic determination of famotidine in plasma. Application to pharmacokinetic study.

A sensitive high-performance liquid chromatographic (HPLC) method for the quantitation of famotidine in human plasma is described. Clopamide was used as the internal standard. Plasma samples were extracted with diethyl ether to eliminate endogenous interferences. Plasma samples were then extracted at alkaline pH with ethyl acetate. Famotidine and the internal standard were readily extracted into the organic solvent. After evaporation of ethyl acetate, the residue was analysed by HPLC. The chromatographic separation was accomplished with an isocratic mobile phase consisting of acetonitrile-water (12:88, v/v) containing 20 mM disodium hydrogenphosphate and 50 mM sodium dodecyl sulphate, adjusted to pH 3. The HPLC microbore column was packed with 5 microns ODS Hypersil. Using ultraviolet detection at 267 nm, the detection limit for plasma famotidine was 5 ng/ml. The calibration curve was linear over the concentration range 5-500 ng/ml. The inter- and intra-assay coefficients of variation were found to be less than 10%. Applicability of the method was demonstrated by a bioavailability/pharmacokinetic study in normal volunteers who received 80 mg famotidine orally.     

Development and validation of a liquid chromatographic method for determination of lacidipine residues on surfaces in the manufacture of pharmaceuticals

A high-performance liquid chromatographic (HPLC) method for the assay of lacidipine residues in swabs collected from various surfaces involved in drug manufacture is described. The swabbing procedure using two cotton swabs was validated applying a wipe test. An RP-HPLC method, developed to determine low quantities of the drug in the presence of its main impurities, was also validated. To remove drug residues from stainless steel and glass surfaces, the first cotton swab must be soaked preferably in acetonitrile whereas, on vinyl surfaces better results are obtained using methanol. The HPLC method selected involves a C12 column, at 40 degrees C, a mixture of acetonitrile-0.05 M ammonium acetate (88:12, v/v) as a mobile phase and UV detection at 282 nm. Recoveries obtained are strongly dependent on the type of surface tested, being higher on stainless steel. The surface material has also different influence on the drug stability. The method was validated over a range of 0.5-100 microg/400 cm2 and had a detection limit of 0.1 microg/400 cm2.     

Indirect resolution of enantiomers of penicillamine by TLC and HPLC using Marfey's reagent and its variants

TLC and HPLC methods were developed for indirect chiral separation of penicillamine (3,3-dimethylcysteine) enantiomers after derivatization with Marfey's reagent (FDNP-Ala-NH(2)) and two of its structural variants, FDNP-Phe-NH(2) and FDNP-Val-NH(2). The binary mobile phase of phenol-water (3:1 v/v) and solvent combinations of acetonitrile and triethylamine phosphate buffer were found to give the best separation in normal and reversed-phase TLC, respectively. The diastereomers were also resolved on a reversed-phase C18 HPLC column with gradient elution of acetonitrile and 0.01 m trifluoroacetic acid. The results due to these three reagents were compared. The method was successful for checking the enantiomeric impurity of l-penicillamine in d-penicillamine and to check the enantiomeric purity of pharmaceutical formulations of d-penicillamine. The method was validated for linearity, repeatability, limit of detection and limit of quantification.     

High-performance liquid chromatography and thin-layer chromatography for the simultaneous quantitation of rabeprazole and mosapride in pharmaceutical products

Simple, sensitive high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) methods are developed for the quantitative estimation of rabeprazole and mosapride in their combined pharmaceutical dosage forms. In HPLC, rabeprazole and mosapride are chromatographed using 0.01M 6.5 pH ammonium acetate buffer-methanol-acetonitrile (40:20:40, v/v, pH 5.70+/-0.02) as the mobile phase at a flow rate of 1.0 mL/min. In TLC, the mobile phase is ethyl acetate-methanol-benzene (2:0.5:2.5, v/v). Both the drugs are scanned at 276 nm. The retention times of rabeprazole and mosapride are found to be 4.93+/-0.01 and 9.79+/-0.02, respectively. The Rf values of rabeprazole and mosapride are found to be 0.42+/-0.02 and 0.61+/-0.02, respectively. The linearities of rabeprazole and mosapride are in the range of 400-2000 ng/mL and 300-1500 ng/mL, respectively, for HPLC; in TLC, the linearities of rabeprazole and mosapride are in the range of 400-1200 ng/spot and 300-900 ng/spot, respectively. The limit of detection is found to be 97.7 ng/mL for rabeprazole and 97.6 ng/mL for mosapride in HPLC; in TLC the limit of detection is found to be 132.29 ng/spot for rabeprazole and 98.25 ng/spot for mosapride. The proposed methods can be applied to the determination of rabeprazole and mosapride in combined pharmaceutical products.     

苦荞叶片中芸香甙的薄层色谱定量研究

根据芸香甙的理化性质，建立了一种测定植物样品中芸香甙含量的薄层紫外扫描法，采用硅胶ＧＦ２５４薄层板，样品经甲醇提取浓缩后点样，展开剂为乙酸乙酯－甲酸－水（８＋１＋１）。用薄层色谱扫描仪在波长３５５ｎｍ处测定芸香甙的吸收强度并直接定量。该法操作简便，灵敏度高，定量准确，结果稳定，应用于苦荞叶片中芸香甙含量测定，得到良好结果，线性范围为０～２．０μｇ／μＬ，斑点的最低检出限为５．０ｎｇ，回收率１０２％