Development and validation of a stability-indicating HPLC method for the determination of degradation products in dipyridamole injection

Summary The development and subsequent validation of an isocratic high-performance liquid chromatographic (HPLC) procedure employing ultraviolet (UV) detection for the determination of degradation products in Dipyridamole Injection is reported. The development of this assay involved the evaluation of several factors including buffer type, ionic strength, pH, organic composition, and column type. The described method is simple, reproducible, accurate, and selective. The precision, relative standard deviation (RSD), amongst five sample preparations for total degradation products was not more than (NMT) 10.2 %, while the individual degradation products were NMT 12.1%. Intermediate precision, as determined from fifteen sample preparations, generated by two Analysts on different HPLC systems over three days, exhibited an RSD for total and individual degradation products of 8.2 % and NMT 27.5 %, respectively. The mean absolute recovery of dipyridamole using the described method is 102.1±1. 9%, (mean±SD, n=12) over the concentration range of 0.03 % to 5.0 % of its label claim of 5 mg mL⁻¹. The limit of detection and limit of quantitation were 0.1 and 0.3 μg mL⁻¹, respectively. The linearity of the peak response was verified with respect to dipyridamole concentration over a range of 0.3 and 50 μg mL⁻¹ (0.03 % to 5.0 % label claim). The Standard and Assay Preparations are stable for up to 48 hours at room temperature. The selectivity was evaluated by subjecting the finished product (Dipyridamole Injection) to thermal, acidic, basic, oxidative and fluorescent radiation stress conditions. No interference in the analysis of degradation products was observed, showing the method is stability-indicating.     

Development and validation of HPLC method for determination of clotrimazole and its two degradation products in spray formulation

A novel simple isocratic HPLC method with UV detection for the determination of three compounds in spray solution (active component clotrimazole and two degradation products imidazole and (2-chlorophenyl)diphenylmethanol) using ibuprofen as an internal standard was developed and validated. The complications with different acido-basic properties of the analysed compounds in HPLC separation - while clotrimazole has pK_a 4.7, imidazole has pK_a 6.9 compared to relatively more acidic (2-chlorophenyl)diphenylmethanol - were finally overcome using a 3.5 μm Zorbax^® SB-Phenyl column (75 mm × 4.6 mm i.d., Agilent Technologies).The optimal mobile phase for separation of clotrimazole, degradation products imidazole and (2-chlorophenyl)diphenylmethanol and ibuprofen as internal standard consists of a mixture of acetonitrile and water (65:35, v/v) with pH* conditioned by phosphoric acid to 3.5. At a flow rate of 0.5 ml min⁻¹ and detection at 210 nm, the total time of analysis was less than 6 min.The method was applied for routine analysis (batch analysis and stability tests) in commercial spray solution.     

Validated reversed phase LC method for quantitative analysis of polymethoxyflavones in citrus peel extracts

Polymethoxyflavones (PMFs), which exist exclusively in the citrus genus, have biological activities including anti-inflammatory, anticarcinogenic, and antiatherogenic properties. A validated RPLC method was developed for quantitative analysis of six major PMFs, namely nobiletin, tangeretin, sinensetin, 5,6,7,4'-tetramethoxyflavone, 3,5,6,7,3',4'-hexamethoxyflavone, and 3,5,6,7,8,3',4'-heptamethoxyflavone. The polar embedded LC stationary phase was able to fully resolve the six analogues. The developed method was fully validated in terms of linearity, accuracy, precision, sensitivity, and system suitability. The LOD of the method was calculated as 0.15 microg/mL and the recovery rate was between 97.0 and 105.1%. This analytical method was successfully applied to quantify the individual PMFs in four commercially available citrus peel extracts (CPEs). Each extract shows significant difference in the PMF composition and concentration. This method may provide a simple, rapid, and reliable tool to help reveal the correlation between the bioactivity of the PMF extracts and the individual PMF content.     

Quantitative reversed phase HPLC analysis of L-ascorbic acid (Vitamin C) and identification of its degradation products

Summary An isocratic ion suppression reversed phase method is utilized for the qualitative and quantitative determination of L-ascorbic acid in fresh fruit juices — complex sample matrices. The selectivity of the method, when a macroreticular poly(styrene-divinylbenzene) reversed phase adsorbent is used, is sufficient to resolve the isomers L-ascorbic acid and D-erythorbic acid (isoascorbic acid) to baseline in under 8 minutes. L-Ascorbic acid solution stability is monitored using the same analytical conditions with the degradation products sufficiently well resolved not to interfere in the quantification of L-ascorbic acid. By the use of commercial materials and the preparation of the proposed products of the oxidative degradation of L-ascorbic acid the degradation sequence and identification of the products in an aged L-ascorbic acid solution is accomplished.     

Development and validation of a reversed phase liquid chromatographic method for separation and determination of related-substances of modafinil in bulk drugs

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method for determination and evaluation of purity of modafinil in bulk drugs using Kromasil C₁₈ column with acetonitrile: 0.02 M ammonium acetate as a mobile phase in gradient elution mode at 30 °C and detection at 225 nm using photodiode array detector has been developed. The effects of pH, temperature and the percent of organic modifier on resolution were studied. Related substances, viz, sulphide, sulphoxide, sulphones of the modafinil, acid and ester derivatives, were separated and quantified. The method was found to be simple, rapid, selective and capable of detecting all process related impurities at trace levels in the finished products of modafinil with detection limits of 0.6-2.4 × 10⁻⁸ g. The method was validated with respect to accuracy, precision, linearity, ruggedness, and limits of detection and quantification. It was found to be suitable not only for monitoring the reactions during the process development but also quality assurance of modafinil.     

Development and validation of SPMMTE HPLC method for analysis of profens from human plasma

A fast, selective and reproducible solid‐phase membrane microtip extraction (SPMMTE) HPLC method has been developed and validated for the analyses of ibuprofen, ketoprofen, and flurbiprofen from human plasma. The analysis was carried out on a C₁₈ (150 × 4.6 mm; 5 μm) column. The mobile phase used was water–acetonitrile (55:45, v/v) adjusted to pH 3.0 using trifluoroacetic acid, at a flow rate 0.5 mL/min with a detection wavelength of 225 nm. The values for the capacity factors for the profen samples ranged from 0.47 to 1.50. The values for the selectivity factor (α) for ketoprofen–flurbiprofen, flurbiprofen–ibuprofen and ibuprofen–ketoprofen combinations from human plasma samples were 1.99, 1.00 and 2.10, respectively. The resolution factors (Rs) for ketoprofen–flurbiprofen, flurbiprofen–ibuprofen and ibuprofen–ketoprofen from plasma samples were 3.00, 1.50 and 4.10, respectively. The percentage recoveries of ibuprofen, ketoprofen, and flurbiprofen from human plasma were 75–85%. All of the profens were separated within 7.0 min, indicating a relatively fast method. During the development of the SPMMTE procedure the parameters of pH, contact time, desorption and types of solvents were optimized. The final method was also found to be efficient, effective and inexpensive. Copyright © 2016 John Wiley & Sons, Ltd.     

Development and validation of a TLC-densitometry method for quantitative analysis of nefopam hydrochloride beside its degradation products

A quantitative densitometric thin-layer chromatographic method for determination of nefopam hydrochloride in pharmaceutical preparations has been established and validated. Nefopam from the formulations was separated and identified on silica gel 60 F₂₅₄ TLC plates with chloroform-methanol-glacial acetic acid (9: 2: 0.1, v/v/v) as mobile phase. Densitometric quantification was performed at absorbance maximum 266 nm. The method was validated for linearity, sensitivity, precision and recovery in accordance with ICH guidelines. The presented method is selective and specific with potential application in pharmaceutical analysis. Nefopam hydrochloride was subjected to acidic and alkaline hydrolysis at different temperatures. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.     

Development and validation of a stability-indicating RP-UPLC method for the quantitative analysis of nabumetone in tablet dosage form

High efficiency and less run time are the basic requirements of high-speed chromatographic separations. To fulfill these requirements, a new separation technique, ultra-performance liquid chromatography (UPLC), has shown promising developments. A rapid, specific, sensitive, and precise reverse-phase UPLC method is developed for the determination of nabumetone in tablet dosage form. In this work, a new isocratic chromatographic method is developed. The newly developed method is applicable for assay determination of the active pharmaceutical ingredient. The chromatographic separation is achieved on a Waters Acquity BEH column (100 mm, i.d., 2.1 mm, 1.7 μm) within a short runtime of 2 min using a mobile phase of 5 mM ammonium acetate-acetonitrile (25:75, v/v), at a flow rate of 0.3 mL/min at an ambient temperature. Quantification is achieved with photodiode array detection at 230 nm, over the concentration range of 0.05-26 μg/mL. Forced degradation studies are also performed for nabumetone bulk drug samples to demonstrate the stability-indicating power of the UPLC method. Comparison of system performance with conventional high-performance liquid chromatography is made with respect to analysis time, efficiency, and sensitivity. The method is validated according to the ICH guidelines and is applied successfully for the determination of nabumetone in tablets.     

Development and validation of a simple and rapid HPLC method for the quantitative determination of voriconazole in rat and beagle dog plasma

A simple and rapid high-performance liquid chromatographic method with UV detection is developed and validated to determine the concentration of voriconazole in rat and beagle dog plasma. After protein precipitation using acetonitrile, the supernatant solution is chromatographed on a Diamonsil C(18) column (250 x 4.6-mm i.d., 5 microm). The mobile phase used is a combination of acetonitrile-water-acetic acid (55:45:0.25, v/v/v) with a pH of 4.0. Detection is achieved by a UV detector monitored at a wavelength of 256 nm. The matrix calibration curves are obtained both in the concentration range of 0.10-50.0 microg/mL in rat and beagle dog plasma, with the lower limit of quantitation of 0.10 microg/mL. The intra- and inter-assay precisions in terms of % relative standard deviation are lower than 8.6% and 6.0% in rat and beagle dog plasma, respectively. The accuracy in terms of % relative error ranged from -0.5% to 8.0% and -0.5% to 6.0% in rat and beagle dog plasma, respectively. This validated method is successfully applied to determine the concentration of voriconazole in plasma after intravenous administration of 36 mg/kg voriconazole to rats and 10 mg/kg voriconazole to beagle dogs, respectively.     

Practical,stable standards for the reversed phase HPLC analysis of peptidic leukotrienes

The stable glutathione derivatives 1, R = n-C₆H₁₃ and R = n-C₈H₁₇, are useful as reference compounds for the identification and quantitation of leukotrienes C₄, D₄ and E₄ by reversed phase HPLC analysis.     

Development and validation of a reversed‐phase HPLC method for licarbazepine monitoring in serum of patients under oxcarbazepine treatment

Licarbazepine is the pharmacologically active metabolite of oxcarbazepine, a drug indicated for the treatment of partial seizures and bipolar disorders. Several HPLC methods have been developed thus far but there is lack of control for interferences from antipsychotic drugs. The aim of the present study was to develop a simple, low‐cost and reliable HPLC‐UV method for the determination of licarbazepine in human serum in the presence of co‐administered antiepileptic, antipsychotic and commonly prescribed drugs. Sample preparation consisted of a single protein precipitation step with methanol. Separation lasted ~9 min on a reversed‐phase C₁₈ column using a mobile phase composed of 50 mm sodium‐dihydrogen‐phosphate‐monohydrate/acetonitrile (70:30, v/v) delivered isocratically at 0.9 mL/min and 30°C. Wavelength was 210 nm and calibration curve was linear with r ² 0.998 over the range 0.2–50.0 μg/mL. Coefficient of variation was <5.03% and bias <−4.92%. Recovery ranged from 99.49 to 104.52% and the limit of detection was 0.0182 μg/mL. No interferences from the matrix or from antiepileptic, antipsychotic and commonly prescribed drugs were observed. The method was applied to serum samples of patients under oxcarbazepine treatment and proved to be a useful tool for the therapeutic drug monitoring of licarbazepine during monotherapy or adjunctive treatment of seizures or affective disorders.     

Development and validation of a high-performance liquid chromatographic method for the analysis of antioxidative phenolic compounds in fennel using a narrow bore reversed phase C18 column

A simple high-performance liquid chromatography (HPLC) method for the separation and quantitative determination of the main antioxidant phenolic compounds from bitter fennel, Foeniculum vulgare, was developed. The use of a narrow bore reversed phase (RP) C₁₈ column and an acidic mobile phase enabled ten compounds (caffeoylquinic acids, dicaffeoylquinic acids, flavonoids and rosmarinic acid) to be separated within a 40 min time analysis. The method was validated to demonstrate its selectivity, linearity, precision, accuracy and robustness. In addition, some parameters were studied to optimize the complete extraction of the phenolic compounds. The method was applied to the evaluation of three different fennel materials: distilled and non-distilled aerial parts, as well as defatted fruits. Distilled fennel was found to contain a higher proportion of antioxidant phenolic compounds than the non-distilled plan material.     

Development and validation of a pre-column reversed phase liquid chromatographic method with fluorescence detection for the determination of primary phenethylamines in dietary supplements and phytoextracts

A sensitive and selective reversed-phase liquid chromatographic (RP-LC) method was developed and validated to determine octopamine, tyramine and Tyrosine (Tyr) in complex matrices as formulations and phytoextracts (Citrus aurantium), after pre-column derivatization with o-phthaldialdehyde (OPA) reagent. The chromatographic separations were performed at room temperature on a Phenomenex Luna C18 column using methanol and sodium acetate buffer (pH 5.5) by varying composition gradient elution as mobile phase and detected flurometrically at λ(em)=455 nm with λ(ex)=340 nm. The results obtained by the proposed method were compared with those achieved by a validated direct RP-LC method with fluorescence detection at λ(em)=310 nm with λ(ex)=275 nm, as reference method, using a Phenomenex Gemini C18 column under isocratic elution conditions with acetonitrile and sodium 1-heptanesulphonate (pH 3), as mobile phase. The higher sensitivity of the derivatization method (detection limit about 0.06 pmol) allowed the sure determination of octopamine present in traces in the examined samples. The repeatability of method (RSD) was ≤1.90% and there was no significant difference between repeatability and intermediate precision data. Recovery studies showed good results 99.5-101.3% with RSD ranging from 0.8 to 1.2%. All analyses were performed by mild conditions in absence of preliminary difficult extraction methodologies or laborious step of sample pre-treatment.     

Development and validation of a reverse-phase liquid chromatographic method for assay and related substances of abacavir sulfate

A simple isocratic liquid chromatographic method was developed for the determination of abacavir from its related substances and assay for the first time. This method involves the usage of C18 (Intertsil octadecyl silane-3V, 150 mm x 4.6 mm, 5 microm) column. The method was validated over the range of 0.002-0.1 mg/mL for chloro impurity, 0.005-0.1 mg/mL for amino impurity and pyrimidine impurity, and 0.005-0.2 mg/mL for abacavir. The mobile phase consists of a mixture of 10 mM ammonium acetate buffer and ACN in the ratio of 90:10. The flow rate was set at 1.0 mL/min with UV detection monitored at 214 nm. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolysis, and thermal degradation. The developed method was validated for linearity, range, precision, accuracy, and specificity. This method can be conveniently used in a quality control laboratory for routine analysis of both related substances and assay.     

Development and optimization of a method for analyzing biodiesel mixtures with non-aqueous reversed phase liquid chromatography

Biodiesel (a mixture of fatty acid esters) is normally analyzed using gas chromatography/flame ionization detection, as specified by the ASTM D6584 and EN14105 standards. This paper proposes a binary gradient method for analyzing biodiesel mixtures using non-aqueous reverse phase HPLC with a UV detector capable of overcoming the drawbacks of the gas chromatographic technique normally used. The new analytical method was developed by means of a statistical sensitivity analysis applied to the main parameters influencing the recording, using the full factorial design method combined with the Yates algorithm and the steepest ascent optimization procedure. The present study shows the influence of the main biodiesel mixture separation analysis parameters. The resulting tool proved valid for analyzing not only biodiesel but also any traces of unreacted oil.     

Development and validation of a HPLC method for determination of levonorgestrel and quinestrol in rat plasma

Levonorgestrel and quinestrol, commonly known as EP‐1, has long been used in the control of wild rodents. Up to the present time, however, no method for simultaneous quantification of levonorgestrel and quinestrol in rat plasma has been reported. In the present study, a sensitive reverse‐phase high‐performance liquid chromatography with ultraviolet detection (RP‐HPLC‐UV) method for quantification of levonorgestrel and quinestrol in rat plasma has been developed. It uses a Kromasil ODS C₁₈ column and acetonitrile‐0.1% formic acid (85 : 15, v/v) mobile phase at ambient temperature. The plasma sample was prepared by hexane–isoamyl alcohol extraction (90 : 10, v/v). The flow rate and detection wavelength were 1.0 mL/min and 230 nm. The correlation coefficients were greater than 0.9995 within 0.08–50 μg/mL for levonorgestrel and 0.12–50 μg/mL for quinestrol, and the limits of detection were 0.02 and 0.05 μg/mL for levonorgestrel and quinestrol, respectively. Average recovery ranged from 92.5 to 96.3% and inter‐day RSDs were less than 7.56%. This method can be applied to the further pharmacokinetic study of levonorgestrel and quinestrol in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of HPLC method for imiquimod determination in skin penetration studies

A simple, rapid and sensitive analytical procedure for the measurement of imiquimod in skin samples after in vitro penetration studies has been developed and validated. In vitro penetration studies were carried out in Franz diffusion cells with porcine skin. Tape stripping technique was used to separate the stratum corneum (SC) from the viable epidermis and dermis. Imiquimod was extracted from skin samples using a 7:3 (v/v) methanol:acetate buffer (100 mM, pH 4.0) solution and ultrasonication. Imiquimod was analyzed by HPLC using C(8) column and UV detection at 242 nm. The mobile phase used was acetonitrile:acetate buffer (pH 4.0, 100 mM):diethylamine (30:69.85:0.15, v/v) with flow rate 1 mL/min. Imiquimod eluted at 4.1 min and the running time was limited to 6.0 min. The procedure was linear across the following concentration ranges: 100-2500 ng/mL for both SC and tape-stripped skin and 20-800 ng/mL for receptor solution. Intra-day and inter-day accuracy and precision values were lower than 20% at the limit of quantitation. The recovery values ranged from 80 to 100%. The method is adequate to assay imiquimod from skin samples, enabling the determination of the cutaneous penetration profile of imiquimod by in vitro studies.     

Development of a novel method combining HPLC fingerprint and multi-ingredients quantitative analysis for quality evaluation of traditional Chinese medicine preparation

A novel method combining high performance liquid chromatography (HPLC) fingerprint and simultanous quantitative analysis of multiple acitve components was developed and validated for quality evaluation of one type of traditional Chinese medicine preparations: Shuang-huang-lian (SHL) oral liquid formulation. For fingerprint analysis, 45 peaks were selected as the common peaks to evaluate the similarities among several different SHL oral liquid preparations collected from manufacturers. Additionally, simultanous quantification of eleven markers, including chlorogenic acid, caffeic acid, rutin, forsythiaside, scutellarin, baicalin, forsythin, luteoloside, apigenin, baicalein and wogonin, was performed. Statistical analysis of the obtained data demonstrated that our method has achieved desired linearity, precision and accuracy. Finally, concentrations of these eleven markers in SHL oral liquid prepared by different manufacturers in China were determined. These results demonstrated that the combination of HPLC chromatographic fingerprint and simultaneous quantification of multi-ingredients offers an efficient and reliable approach for quality evaluation of SHL oral liquid preparations.