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

A thin-layer chromatographic (TLC) method with densitometric quantitation using the absorption reflectance mode at 280 nm was developed and validated for the determination of chloramphenicol residues in controlling pharmaceutical equipment cleanliness. Simulated samples at residue levels 0.5, 1, and 1.2 mg/m2 were prepared by spreading the calculated amount of chloramphenicol solution on a 10 dm2 stainless steel surface. After evaporation of the solvent, the residue was removed by 2 methanol-wetted cotton swabs, which were then extracted with methanol. The extract was applied on a high-performance TLC (HPTLC) silica gel F254 plate together with standards ranging from 10 to 60 ng. Plates were developed in a horizontal developing chamber from both sides (36 applications per plate) by using n-hexane-ethyl acetate (35 + 65, v/v) as developing solvent. The mean recovery (n=6) at 1 mg/m2 was 95.8%, and the coefficient of variation was 5.8%. The absolute detection limit was 3 ng, and the quantitation limit 10 ng. The method detection limit was 0.3 mg/m2 by swabbing 2.5 dm2 and 0.075 mg/m2 by swabbing 10 dm2. Chloramphenicol was stable on the plate 2 h before and 24 h after development. Additionally, it was stable during 7 days storage on the cotton swabs in the solvent at room temperature and in diluted standard solution stored in darkness at 4 degrees C. The method can be applied to routine control of pharmaceutical equipment cleanliness by sampling from the stainless steel surface areas of 2.5 to 10 dm2, and an acceptable residue limit of 1 mg/m2.     

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.     

High-performance thin-layer chromatography method for monitoring norfloxacin residues on pharmaceutical equipment surfaces

This paper presents a high-performance thin-layer chromatography (HPTLC) method with direct fluorescence measurement for the determination of norfloxacin. The method was validated for the monitoring of norfloxacin residues on stainless steel surfaces at the allowed limit of 10 mg of norfloxacin per square meter. However, it can be adapted for lower amounts of residues owing to the low detection limit of norfloxacin (about 5 ng) and can also be used for other surface materials. Test solutions were analyzed by the new HPTLC method and the known HPLC method for comparison. Accuracy and precision of the new HPTLC method, with a subsequent quantification by densitometer or video system, are comparable with those of the HPLC method.     

Determination of pentoxifylline in pharmaceutical formulations using iodine as oxidizing agent

Simple, selective and sensitive spectrophotometric methods are described for the determination of pentoxifylline, based on the haloform reaction with a known and excess of standard iodine solution under alkaline conditions. The excess of iodine is determined at pH 3.0 with metol–isoniazid (λ_max=620 nm; method A) or wool fast blue BL (λ_max=540 nm, method B). All the variables have been optimised and the reaction mechanisms presented. Regression analysis of Beer's law plots showed good correlation in the concentration ranges 4.0–24.0 and 0.4–2.4 mg ml⁻¹ for methods A and B respectively. No interferences were observed from excipients and the validity of the methods was tested by analysing pharmaceutical formulations. Recoveries were 99.0–100.0%. The concentration measurements were reproducible within a relative standard deviation of 1.0%.     

Multicomponent pharmaceutical cocrystals: furosemide and pentoxifylline

The combination of the active pharmaceutical ingredients furosemide [4‐chloro‐2‐(furan‐2‐ylmethylamino)‐5‐sulfamoylbenzoic acid] and pentoxifylline [3,7‐dimethyl‐1‐(5‐oxohexyl)‐3,7‐dihydro‐1H‐purine‐2,6‐dione] produces a 1:1 cocrystal, C₁₂H₁₁ClN₂O₅S·C₁₃H₁₈N₄O₃, (I), a 1:1 cocrystal hydrate, C₁₂H₁₁ClN₂O₅S·C₁₃H₁₈N₄O₃·H₂O, (II), and a 1:1 cocrystal acetone solvate, C₁₂H₁₁ClN₂O₅S·C₁₃H₁₈N₄O₃·C₂H₆O, (III). These structures exhibit the presence of a rarely encountered synthon with the graph set R₂²(7). All potential hydrogen‐bond donors of furosemide participate in hydrogen‐bond formation in (I)–(III). However, only two hydrogen‐bond acceptors of furosemide are active in (I) and (II), and only one is active in (III). Four hydrogen‐bond acceptors of pentoxifylline are active in (II), three in (I) and two in (III). These observations are in good agreement with the calculated packing indexes of 69.5, 69.6 and 68.8% for (II), (I) and (III), respectively.     

Simultaneous determination of fluocortolone, fluocortolone caproate and nicotinic acid benzylester in pharmaceutical preparations by TLC

Summary A direct quantitative thin-layer Chromatographic method for the determination of fluocortolone, fluocortolone caproate and nicotinic acid benzylester in the presence of each other in pharmaceutical preparations is described. The active ingredients were extracted from the preparations with an electronically controlled extraction apparatus within 15 min. Development of thin-layer chromatograms was carried out on silica gel 60 F254 precoated plates. All three active ingredients can be separated on one plate using one solvent system namely diethyl ether — hexane — benzene (75251) and determined directly by the remission method using a densitometer. Fluocortolone and fluocortolone caproate were measured at 243 nm and nicotinic acid benzylester at 263 nm. Evaluation of thinlayer chromatograms takes place on-line from the linear calibration curves using an IBM 1800 computer. The described method is suitable for analyses of these substances in pharmaceutical preparations, such as ointments and creams and can be well reproduced with a coefficient of variation between 1.3–3.5% for the three active ingredients.

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Gleichzeitige Bestimmung von Fluocortolon, Fluocortoloncaproat und Nicotinsäurebenzylester in pharmazeutischen Zubereitungen durch DC

     

Membrane distillation as an online concentration technique: application to the determination of pharmaceutical residues in natural waters

Membrane distillation (MD) is presented for the first time as a real-time, online concentration technique, where the aqueous matrix is removed from the sample to enhance analyte enrichment. Therefore, MD is a universal method for a wide range of compounds and is unlike conventional membrane extractions that rely on the permeation of the solute into an extractant phase. The MD process showed excellent precision with relative standard deviation between 3% and 5%, linear calibration, and the detection limits for pharmaceutical compounds in the range of 0.01 to 20 mg L⁻¹ by HPLC-UV analysis. The temperature and flow rate of the feed solution were found to be important variables.     

Stability indicating method for the determination of paracetamol in its pharmaceutical preparations by TLC densitometric method

Simple, sensitive and accurate thin layer procedure was described for a quantitative determination of paracetamol in its bulk powder and in its pharmaceutical dosage forms in the presence of its degradation product. The method consists of dissolving the drug in methanol and then spotting the solution on a thin layer of silica gel G254. Paracetamol was separated on silica gel using the mixture of the mobile phase, ethyl acetate: benzene: acetic acid in a ratio (1:1:0.05 v/v/v).Absorbance measurements (detection of reflectance) of the separated drug were carried out at 250 nm. Calibration curves were established in the concentration range of 5–20 mcg/spot for paracetamol. Quantitation is achieved by comparing the area under the peaks obtained from scanning the thin layer chromatographic plates in a spectrodensitometer. The method has been successfully applied to pharmaceutical preparations (capsules) and the results obtained were statistically compared with those obtained by applying the reference method.     

Trace-level determination of pharmaceutical residues by LC-MS/MS in natural and treated waters. A pilot-survey study

A pilot-survey study was performed by collecting samples (influent and effluent wastewaters, rivers and tap waters) from different locations in Europe (Spain, Belgium, Germany and Slovenia). A solid-phase extraction (SPE) followed by liquid chromatography–tandem mass spectrometry method was applied for the determination of pharmaceuticals (ibuprofen, naproxen, ketoprofen, diclofenac and clofibric acid). Method detection limits and method quantification limits were at the parts-per-trillion level (7.5–75 ng/L). The recovery rates of the SPE from deionized water and effluent wastewater samples spiked at 100- and 1,000-ng/L levels ranged from 87 to 95%. Identification criteria in compliance with the EU regulation for confirmatory methods of organic residues were applied. A detailed study of signal suppression evaluation for analysis of pharmaceutical residues in effluent wastewaters is presented.     

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.     

Immobilization of antibodies on glass surfaces through sugar residues

This work characterizes substrates for immunoassays obtained through the immobilization of vectorially oriented antibodies on glass. The method of preparation is based on the condensation reaction between an aldehyde group on the F(c) portion of antibodies and the hydrazide group on the modified glass surface. Light microscopy and AFM imaging in height and phase modes were used to assess the properties of the modified surface. Both techniques are consistent with a fairly uniform antibody coverage on the micrometer and submicrometer scales. ELISA tests were used to evaluate the activity and surface distribution of immobilized antibodies as well as nonspecific binding to surfaces after various modification steps. It was shown that exposure of the surfaces to a BSA solution minimized nonspecific binding to undetectable levels.     

Validation of the removal of acetylsalicylic acid. Recovery and determination of residues on various surfaces by high performance liquid chromatographic

The validation of a procedure to clean glass, vinyl and stainless steel surfaces that have been exposed to acetylsalicylic acid during its manufacture is described. The cleaning procedure using two cotton swabs moistened with the mobile phase was validated using a wipe-test and a high-performance liquid chromatography (HPLC) method developed to determine low quantities of the acid. The HPLC method involves an octadecylsilane column at 55°C, a mixture of water–acetonitrile–orthophosphoric acid (779:220:1, v/v) as mobile phase and detection at 226 nm. Recoveries of 86%, 90% and 94% were obtained from vinyl, glass and stainless steel plates respectively. The validation gave acceptable levels of sensitivity, recovery, precision and linearity.     

Stability indicating HPTLC determination of clopidogrel bisulphate as bulk drug and in pharmaceutical dosage form

A sensitive, selective, precise and stability indicating high-performance thin layer chromatographic method of analysis of clopidogrel bisulphate both as a bulk drug and in formulations was developed and validated in pharmaceutical dosage form. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of carbon tetrachloride-chloroform-acetone (6:4:0.15, v/v/v). This system was found to give compact spots for clopidogrel bisulphate (R_f value of 0.30±0.01). Clopidogrel bisulphate was subjected to acid and alkali hydrolysis, oxidation, photodegradation and dry heat treatment. Also the degraded products were well separated from the pure drug. Densitometric analysis of clopidogrel bisulphate was carried out in the absorbance mode at 230 nm. The linear regression data for the calibration plots showed good linear relationship with r²=0.999±0.001 in the concentration range of 200-1000 ng. The mean value of correlation coefficient, slope and intercept were 0.999±0.001, 0.093±0.011 and 8.83±0.99, respectively. The method was validated for precision, accuracy, ruggedness and recovery. The limits of detection and quantitation were 40 and 120 ng per spot, respectively. The drug undergoes degradation under acidic and basic conditions, oxidation and dry heat treatment. All the peaks of degraded product were resolved from the standard drug with significantly different R_f values. This indicates that the drug is susceptible to acid-base hydrolysis, oxidation and dry heat degradation. Statistical analysis proves that the method is reproducible and selective for the estimation of the said drug. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.     

Colorimetric determination of isoniazid and its pharmaceutical formulations

Two colorimetric methods for the estimation of isoniazid are developed. The first method depends on coupling of isoniazid with diazotized 1-amino anthraquinone zinc chloride salt (fast red AL salt) to form a red colour (lambda(max) 510 nm). The second one is based on the formation of a green complex (lambda(max) 655 nm) between the acid hydrazide and 2,6-dimethoxy-1,4-benzoquinone (DMBQ). All measurements of the two procedures were carried out in the presence of sodium hydroxide at room temperature (20 +/- 3 degrees C). The two methods are applied for the determination of isoniazid in presence of congenial drugs, vitamins and additives normally encountered with it in pharmaceutical dosage forms. The reliability of these methods was established by parallel determination with the reported and official methods.     

Extraction and determination of zinc in pharmaceutical samples

A systematic study of extraction of zinc salicylate is reported. Optimum conditions for the extraction and determination of zinc are evaluated from a critical study of the effect of pH, sodium salicylate concentration and triphenylphosphine oxide concentration. The effect of foreign ions on the extraction is also discussed. The probable composition of the species has been deduced from the extraction data. The method has been used to separate zinc from cadmium and mercury in binary mixtures and for the determination of zinc in various pharmaceutical products.     

Use of automated solid-phase extraction equipment for the determination of ivermectin residues in animal liver by HPLC.

An improvement of the method commonly used for the determination of ivermectin is proposed. An automatic system is used for the solid-phase extraction column purification stage in order to provide more efficient and fast sample preparation, prior to separation and quantification by HPLC. Some tests were performed to obtain recovery and repeatability data. The mean recovery for spiked samples (five replicates twice at three concentration levels) was more than 90% in the concentration range 7.5-30 ng g-1. The tests for accuracy (five replicates at three concentration levels) gave a standard error of -2.0% at the highest and -19.2% at the lowest concentration. Concerning repeatability data (five replicates twice at three concentration levels), in the concentration range 7.5-30 ng g-1 the RSD varied from 9.29 to 2.15%. The described method was used in a survey on the presence of ivermectin in food-producing animals in two regions of southern Italy; no positives out of 250 liver samples were found.     

Quantitative determination of iron and copper in cotton material by TLC

Summary A TLC method for the separation of quantitative determination of copper and iron in cotton material is described. The optimal solvent system is 9:2:1 (v/v), ethanol-nitric acid-hydrochloric acid and the locating reagent is Na-diethyldithiocarbamate. Regression analysis shows that the most precise results can be obtained for the concentration range of 0.0400–0.0800 mg/ml of iron and copper.