Development of quantitative HPTLC-densitometry methods following a model approach for transfer of TLC screening methods for pharmaceutical products of atenolol,chloramphenicol, furosemide,glibenclamide, penicillin V potassium,and praziquantel

Transfer of six thin-layer chromatography (TLC) Global Pharma Health Fund E.V. Minilab manual protocols for detecting fake drugs in pharmaceutical products to quantitative high-performance TLC (HPTLC)-densitometry methods was performed following a previously published model process. The developed and validated methods for tablets or capsules containing atenolol, chloramphenicol, furosemide, glibenclamide, penicillin V potassium, and praziquantel involved use of a limited list of inexpensive, relatively nontoxic, readily available solvents and other reagents; silica gel 60?F₂₅₄ plates; automated bandwise sample and standard solution application; ascending mobile phase development of plates in a chamber; and automated slit scanning densitometry for detection, identification, and quantification. Validation data for methods developed in an early version of the transfer model process that did not include standard addition validation are reported for pharmaceutical products containing amitriptyline HCl, amodiaquine, diphenhydramine HCl, and mebendazole.     

Development and validation of a generic RP‐HPLC PDA method for the simultaneous separation and quantification of active ingredients in cold and cough medicines

A novel generic reverse phase high performance liquid chromatography (RP‐HPLC) method is developed and validated for simultaneous determination of seven pharmaceutically active ingredients, namely, acetaminophen, dextromethorphan, doxylamine, phenylephrine, guaifenesin, caffeine and aspirin. All seven ingredients were quantified in soft gel, syrup and tablet formulations of the over‐the‐counter US‐marketed products, as per the guidelines of the International Conference on Harmonization. The separation was achieved in a 16 min run time on an Agilent Zorbax Phenyl column using a gradient method with two mobile phases. Mobile phase A was 0.15% trifluoro acetic acid in purified water and while mobile phase B was a mixture of acetonitrile and methanol (750:250 v/v) with 0.02% trifluoro acetic acid. The flow rate was 1.0 mL min^?1 and injection volume was 10 μL. Detection was performed at 280 nm using a photodiode array detector. As part of the method validation, specificity, linearity, precision and recovery parameters were verified. The concentration and area relationships were linear (R² > 0.999), over the concentration ranges 20–120 μg mL^?1 for acetaminophen, 75–450 μg mL^?1 for dextromethorphan, 31.25–187.5 μg mL^?1 for doxylamine, 25–150 μg mL^?1 for phenylephrine, 25–150 μg mL^?1 for aspirin, 6.5–39 μg mL^?1 for caffeine and 12–72 μg mL^?1 for guaifenesin. The relative standard deviations for precision and intermediate precision were <1.5%. The proposed RP‐HPLC generic method is applicable for routine analysis of cold and cough over‐the‐counter products.     

Development and validation of an MEKC method for determination of nitrogen-containing drugs in pharmaceutical preparations

A fast and accurate micellar electrokinetic capillary chromatography method was developed for quality control of pharmaceutical preparations containing cold remedies as acetaminophen, salicylamide, caffeine, phenylephrine, pseudoephedrine, norephedrine and chlorpheniramine. The method optimization was realized on a Beckman P/ACE System MDQ instrument. The baseline separation of seven analytes was performed in an uncoated fused silica capillary internal diameter (ID)=50 microm using tris-borate (20 mM, pH=8.5) containing sodium dodecyl sulphate 30 mM BGE. On line-UV detection at 214 nm was performed and the applied voltage was 10 kV. The operating temperature was 25 degrees C. After experimental conditions optimization, the proposed method was validated. The evaluated parameters were: precision of migration time and of corrected peak area ratio, linearity range, limit of detection, limit of quantification, accuracy (recovery), ruggedness and applicability. The method was then successfully applied for the analysis of three pharmaceutical preparations containing some of the analytes listed before.     

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.     

Simple HPLC method for simultaneous determination of acetaminophen, caffeine and chlorpheniramine maleate in tablet formulations

Summary A simple, rapid and accurate, routine-HPLC method is described for simultaneous determination of acetaminophen, caffeine and chlorpheniramine maleate in a new tablet formulation Chromatographic separation of the three pharmaceuticals was achieved on a Hypersil CN column (150×5.0 mm, 5 μm) using a mobile phase comprising a mixture of acetonitrile, an ion-pair solution and tetrahydrofuran (13:14:87, v/v,pH4.5). The flow-rate was changed from 1.0 mL min⁻¹ (in 0≈7.5 min) to 1.8 mL min⁻¹ (after 3.5 min). was complete in <10 min. The method was validated for system suitability, linearity, accuracy, precision, limits of detection and quantitation, and robustness. Linearity, accuracy and precision were found to be acceptable over the ranges 31.6≈315.8 μg mL⁻¹ for acetaminophen, 9.5≈94.6 μg mL⁻¹ for caffeine and 1.4≈13.8 μg mL⁻¹ for chlorpheniramine maleate.     

Improved separation and quantitative determination of hydrocarbon types in gas oil by normal phase high‐performance TLC with UV and fluorescence scanning densitometry

Improved methods for separation and quantitative determination of hydrocarbon types from gas oil have been developed, which were based on high‐performance thin‐layer chromatography with ultraviolet and fluorescence scanning densitometry using horizontal elution. One of the methods allows the separation, detection, and determination of alkanes and naphthenes to be carried out, using berberine‐impregnated silica gel HPTLC plates, elution with n‐hexane, and berberine‐induced fluorescence detection at 365 nm. Another developed method allows total aromatics to be determined using silica gel HPTLC plates by elution with n‐hexane and acetone, and UV detection. In turn, PACs over three aromatic rings can be determined on either silica gel or caffeine‐impregnated silica gel HPTLC plates, elution with n‐hexane, and selective detection using native fluorescence at 365 nm. Concentrations lower than 5 wt% can be determined using this technique. In addition, a technique for an efficient, baseline‐resolved separation of a gas oil according to the number of aromatics rings (mono + di‐, tri‐, and polyaromatic compounds with more than three rings) is presented here. This technique involves a multistep elution on a mixed (silica gel and caffeine‐impregnated silica gel HPTLC plate) using a counter‐elution device, and UV detection.     

Development and validation of a capillary zone electrophoretic method for the determination of bisphosphonate and phosphonate impurities in clodronate

Capillary zone electrophoresis with direct UV detection at low wavelength and reversed polarity was applied for the separation and quantitation of bisphosphonate and phosphonate impurities in clodronate bulk material. Polyacrylamide-coated capillaries were used to reduce the interactions between the analytes and the electric double layer of the capillary, and to minimize electroosmotic flow. Study was made of the major factors affecting the separation, i.e., pH and ionic strength of the electrolyte solution and various instrumental parameters. The developed method provided reproducible separations of clodronate and related impurities (between-day precision of migration times: RSD < 2.3%, 275 runs). Acceptable validation results in the impurity quantitation range of 0.5-7.5 microg ml(-1) (corresponding to 0.1-1.5% of clodronate working concentration) were obtained in specificity, within-day and between-day precision, accuracy and linearity.     

Comparison between capillary electrophoresis and liquid chromatography for the determination of diclofenac sodium in a pharmaceutical tablet

Two novel analytical methodologies using capillary electrophoresis (CE) and liquid chromatography (LC) were developed and compared for the determination of diclofenac sodium in commercial and simulated tablet formulations. The CE analysis was performed in a bare fused-silica capillary with 75 microm id and total length of 50 cm (28 cm to the detector) with a buffer solution of 20 mM sodium tetraborate, pH 9.23. The applied voltage was 20 kV, and acetaminophen was used as the internal standard (IS). The LC analysis was performed with a LiChrospher 100 RP-18 (5 microm) column and a mobile phase of methanol-diluted glacial acetic acid (0.3 parts in 2500; 75 + 25) at a flow rate of 0.9 mL/min with propylparaben as the IS. In both analyses, detection was by ultraviolet absorption at 276 nm. Under optimized conditions, the CE migration times for the diclofenac sodium standard and acetaminophen (IS) were 2.07 and 1.59 min, respectively, and the LC retention times for the diclofenac sodium standard and propylparaben (IS) were 3.98 and 2.26 min, respectively. The resolution and efficiency for CE were 14.2 and 1.6 x 10(5) plates/m, respectively, and for LC, 5.0 and 8.6 x 10(3) plates/m, respectively. Calibration curves of peak area versus concentration gave correlation coefficients of 0.9992 for CE and 0.9994 for LC. The limits of detection and quantitation were 8.40 and 25.46 microg/mL, respectively, for CE and 4.60 and 13.93 microg/mL, respectively, for LC. Coefficients of variation were 1.68 and 0.37% for CE and LC, respectively. Average recoveries obtained with CE and LC were 103.12+/-0.90 and 99.59+/-0.21%, respectively. Although both methodologies were shown to be suitable for the determination of diclofenac sodium in tablets, performing in a similar manner with regard to several aspects (linearity, recovery, and specificity), CE provided faster analysis and better column efficiency, whereas LC provided superior repeatability and sensitivity.     

Simultaneous Determination of Acetaminophen, Caffeine, and Chlorphenamine Maleate in Paracetamol and Chlorphenamine Maleate Granules

A simple and rapid HPLC method using phenacetin (PHN) as internal standard has been developed for simultaneous determination of acetaminophen, caffeine, and chlorphenamine maleate in the product compound paracetamol and chlorphenamine maleate granules. Separation and quantitation were achieved on a 250 mm × 4.6 mm, 5 μm particle, C₁₈ column. The mobile phase was methanol 0.05 mol L^?1 aqueous KH₂PO₄ solution, 45:55 (v/v), containing 0.1% triethylamine and adjusted to pH 3.6 by addition of phosphoric acid; the flow rate was 1.0 mL min^?1. Detection of all compounds was by UV absorbance at 260 nm and elution of the analytes was achieved in less than 12 min. The linearity, accuracy, and precision of the method were acceptable to good over the concentration ranges 6.4–153.6 μg mL^?1 for acetaminophen, 5.0–120.0 μg mL^?1 for caffeine, and 9.6–230.4 μg mL^?1 for chlorphenamine maleate.     

Stability‐indicating HPLC method for simultaneous quantification of 14 impurities in excedrin tablet formulations and identification of new impurity by LC–MS in accelerated stability studies

We developed novel stability‐indicating HPLC method for simultaneous estimation of 14 impurities in excedrin tablet, a formulation with a combination of acetaminophen, aspirin, and caffeine. In addition, a new impurity that was generated through degradation of aspirin at high temperatures during the accelerated stability conditions was positively identified and confirmed, using liquid chromatography–mass spectrometry technique. The HPLC method was optimized using the Inertsustain C₁₈, 250 × 4.6 mm, 5.0 μm column, employing simple gradient method. Forced degradation studies were performed under acidic, basic, oxidative and thermal conditions to prove the scope and stability‐indicating the nature of the method. The optimized method was validated as per the International Conference on Harmonization guidelines. The HPLC method showed linearity from LOQ concentration to 21 μg mL^?1. Precision and intermediate precision values were <5% RSD. The validated HPLC method is currently applied for the routine testing of excedrin tablet formulations in quality control laboratories.     

Thin-layer chromatographic separation and in situ fluorimetric determination of ofloxacin in plasma and pleural fluid

A thin-layer chromatographic assay for the determination of ofloxacin in human plasma and pleural fluid is described. After extraction of ofloxacin from samples with dichloromethane, chromatography was performed on thin-layer plates (silica gel) with a mobile phase consisting of ethanol and water; the tank atmosphere was equilibrated with concentrated ammonia. The precision of the assay could be considerably increased along with the measured fluorescence intensity of ofloxacin by spraying the plate with a citric acid solution and dipping it into paraffin or using a mixture of both components. Peaks were quantified by densitometric evaluation of the chromatograms. The method shows a very low limit of detection (1 ng/ml) as well as good precision and linearity in the range 0.001-2.0 micrograms/ml for both plasma and pleural fluid.     

在线扫集-胶束毛细管电动色谱法测定复方氨酚烷胺胶囊中的3种有效成分

以在线扫集-胶束毛细管电动色谱法(Sweeping-MEKC)测定了复方氨酚烷胺胶囊中的马来酸氯苯那敏、咖啡因和对乙酰氨基酚3种有效成分。考察了缓冲溶液pH值、SDS浓度、分离电压及进样时间等对分离效果的影响。优化条件:以未涂层熔融石英毛细管(55 cm×50μm,有效柱长35 cm)为分离柱;环境温度25℃;80 mmol/L十二烷基磺酸钠+20 mmol/L NaH2PO4(pH 2.2)+15%乙腈为缓冲体系,分离电压-20kV,进样时间60 s(H=20.0 cm),测量波长210 nm。在该条件下氯苯那敏、咖啡因和对乙酰氨基酚在25min内出峰,峰面积RSD均小于4%;线性范围分别为2.45～39.17、1.61～25.76、1.58～25.28 mg/L;检出限(S/N=3)分别达139、34、24μg/L,回收率分别为96%～101%、98%～102%、96%～102%。     

Development and validation of an analytical method for metformin hydrochloride and its related compound (1-cyanoguanidine) in tablet formulations by HPLC-UV

A simple, and stability-indicating liquid chromatographic method was developed and validated for the analysis of metformin hydrochloride and its related compound (1-cyanoguanidine) in tablet formulations. Liquid chromatography with a UV detector at a wavelength of 232 nm using a Nova-Pak silica column was employed in this study. Isocratic elution was employed using a mixture of ammonium dihydrogen phosphate buffer and methanol (21:79, v/v). This new method was validated in accordance with USP requirements for new methods for assay determination, which include accuracy, precision, specificity, linearity and range. The current method demonstrates good linearity over the range of 0.01-0.03 mg mL⁻¹ of metformin hydrochloride. The accuracy of the method is 100.4%. The precision of this method reflected by relative standard deviation of replicates is 0.30%. Validation of the same method for 1-cyanoguanidine determination was also performed according to USP requirements for quantitative determination of impurities which include accuracy, precision, linearity and range, selectivity, and limit of quantification (LOQ). Low LOQ of 1-cyanoguanidine using this method enables the detection and quantification of this impurity at low concentration.     

Simultaneous determination of caffeine, ergotamine, and metamizol in solid pharmaceutical formulation by HPTLC-UV-FLD with mass confirmation by online HPTLC-ESI-MS

A new high-throughput method is developed to quantify caffeine, ergotamine, and metamizol in a solid pharmaceutical formulation. After dissolution, the compounds are separated on silica gel 60 F(254) high-performance thin-layer chromatography (HPTLC) plates with ethyl acetate-methanol-ammonia 90:15:1 (v/v/v) as the mobile phase. Detection is performed by UV absorption at 274 nm for caffeine and metamizol, and by fluorescence at 313 /> 340 nm for ergotamine. Calibrations are linear or polynomial with determination coefficients (R(2)) >or= 0.9986. Recoveries of the three compounds are between 95% and 102% at three different concentration levels. Repeatability [relative standard deviation (RSD)] of all substances in the matrix is between +/- 0.9% and +/- 1.7%. Intermediate precision (RSD) of the three compounds range from +/- 2.0% to +/- 3.1%. Mass confirmation is performed by a single quadrupole mass spectrometry in positive electrospray ionization full scan mode for caffeine and ergotamine and in negative mode for metamizol. The results proved that this method is a simple and reliable alternative for routine analysis.     

Solid-phase extraction and HPTLC determination of isoniazid and acetylisoniazid in serum. Comparison with HPLC

A new solid-phase extraction (SPE) and quantitative determination of isoniazid (INH) and its acetyl metabolite (AcINH) in serum by high-performance thin-layer chromatography (HPTLC) is presented. Alkalized serum samples with nicotinamide as an internal standard are applied to an SPE cartridge containing a new SPE sorbent, [poly (divinylbenzene-co-N-vinylpyrrolidone )]. A simple procedure of conditioning, washing, and eluting steps is described. After evaporation of the eluates to dryness and reconstitution, one-dimensional HPTLC is performed on silica gel plates with ethyl acetate-methanol (70:30) as a mobile phase. Quantitation is done by densitometry. Convenient validation parameters are obtained (linearity, limits of detection and quantitation, precision, and accuracy) for INH and AcINH. The method is compared with a high-performance liquid chromatographic (HPLC) technique developed in the laboratory, and satisfactory correlation is found between data from the two techniques. The HPTLC method is sensitive and specific and is used to quantitate INH and AcINH in patient blood serum, and the results are compared with those obtained by HPLC.     

Preconcentration and determination of mercury(II) at a chemically modified electrode containing 3-(2-thioimidazolyl)propyl silica gel.

A mercury-sensitive chemically modified graphite paste electrode was constructed by incorporating modified silica gel into a conventional graphite paste electrode. The functional group attached to the (3-chloropropyl) silica gel surface was 2-mercaptoimidazole, giving a new product denoted by 3-(2-thioimidazolyl)propyl silica gel, which is able to complex mercury ions. Mercury was chemically adsorbed on the modified graphite paste electrode containing 3-(2-thioimidazolyl)propyl silica (TIPSG GPE) by immersion in a Hg(II) solution, and the resultant surface was characterized by cyclic and differential pulse anodic stripping voltammetry. One cathodic peak at 0.1 V and other anodic peak at 0.34 V were observed on scanning the potential from -0.1 to 0.8 V (0.01 M KNO3; v = 2.0 mV s(-1) vs. Ag/AgCl). The anodic peak at 0.34 V show an excellent sensitivity for Hg(II) ions in the presence of several foreign ions. A calibration graph covering the concentration range from 0.02 to 2 mg L(-1) was obtained. The detection limit was estimated to be 5 microg L(-1). The precision for six determinations of 0.05 and 0.26 mg L(-1) Hg(II) was 3.0 and 2.5% (relative standard deviation), respectively. The method can be used to determine the concentration of mercury(II) in natural waters contaminated by this metal.     

Instrumental planar chromatographic method for determination of carbamazepine in human serum

An instrumental planar chromatographic (HPTLC) method for quantification of carbamazepine in human serum was developed using liquid‐liquid extraction with dichloromethane, fluorescence activation with perchloric acid 60%/ethanol/water (1:1:1, v/v) and fluorescence detection. Planar chromatographic separation was performed on precoated silica gel F254 HPTLC plates using a mixture of ethyl acetate/toluene/methanol/acetic acid glacial (5:4:0.5:0.5, v/v) as mobile phase. Densitometric detection was done at 366 nm. The method was validated for linearity, precision and accuracy. Linear calibration curves in the range of 3 and 20 ng/μL showed correlation coefficient of 0.998. The intra‐assay and inter‐assay precision, expressed as the RSD, were in the range of 0.41–1.24% (n = 3) and 2.17–3.17% (n = 9), respectively. The LOD was 0.19 ng, and the LOQ was 0.57 ng. Accuracy, calculated as percentage recovery, was between 98.98 and 101.96%, with a RSD not higher than 1.52%. The method was selective for the active principle tested. In conclusion, the method is useful for quantitative determination of carbamazepine in human serum.     

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.     

Simultaneous determination of atenolol and amiloride in pharmaceutical preparations by capillary zone electrophoresis with capacitively coupled contactless conductivity detection

Capillary zone electrophoresis coupled with a capacitively coupled contactless conductivity detector (CE‐C⁴D) has been employed for the determination of atenolol and amiloride in pharmaceutical formulations. Acetic acid (150 mm ) was used as background electrolyte. The influence of several factors (detector excitation voltage and frequency, buffer concentration, applied voltage, capillary temperature and injection time) was studied. Non‐UV‐absorbing L‐valine was used as internal standard; the analytes were all separated in less than 7 min. The separation was carried out in normal polarity mode at 28°C, 25 kV and using hydrodynamic injection (25 s). The separation was effected in an uncoated fused‐silica capillary (75 μm, i.d. × 52 cm). The CE‐C⁴D method was validated with respect to linearity, limit of detection and quantification, accuracy, precision and selectivity. Calibration curves were linear over the range 5–250 μg/mL for the studied analytes. The relative standard deviations of intra‐ and inter‐day migration times and corrected peak areas were less than 6.0%. The method showed good precision and accuracy and was successfully applied to the simultaneous determination of atenolol and amiloride in different pharmaceutical tablet formulations. Copyright © 2010 John Wiley & Sons, Ltd.     

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.