Method development and validation for the GC-FID assay of p-cymene in tea tree oil formulation

This paper describes the development and validation of an isothermal gas chromatography-flame ionisation detection (GC-FID) method for the assay of pure tea tree oil. The chromatographic conditions of the method employ a 5% carbowax packed column (20 m x 0.25 mm), isothermal elution with hydrogen at a column flow of 36 ml/min, injector and detector temperature at 220 degrees C and oven temperature at 100 degrees C, and a 1.5 microl injection volume. Samples and standard were diluted in hexane. The calibration curve for p-cymene was linear (r2=0.9995) from 20 to 120% range of the analytical concentration of 100 microg/ml. The precision of this method was calculated as the relative standard deviation (R.S.D.) was 0.66% (n=6). The R.S.D. for intermediate precision study was 0.13 and recovery of the p-cymene ranged between 93.39 and 97.86%. The limits of detection and quantitation were determined to be 2.08 and 10.39 ng/ml, respectively.     

Method development and validation for the HPLC potency assay of troglitazone tablets

This paper describes the development and validation of an isocratic, reversed-phase, high performance liquid chromatographic (HPLC) method for the assay of 200-mg troglitazone tablets. The chromatographic conditions of the method employ a YMC ODS-A, 120 A (4.6 x 150 mm, 5 microm) column, isocratic elution with (50 mM aqueous NaH2PO4, pH 4.0):acetonitrile:methanol, (35:50:15, v/v/v) as the mobile phase at a flow rate of 1.0 ml/min, a 10 microl injection volume, and ulltraviolet (UV) detection at 225 nm. The active was analyzed at ambient column temperature, using peak area responses.     

Method development and validation for the HPLC assay (potency and related substances) for 20 mg paroxetine tablets

A reversed phase high performance liquid chromatographic (HPLC) method was developed and validated for use as a stability indicating assay (potency and related substances) of paroxetine in paroxetine hydrochloride 20 mg tablets. Assay samples were extracted at a paroxetine concentration of 0.4 mg ml(-1) utilizing mobile phase as the extraction solvent. The chromatographic conditions employed a C18 column (Inertsil, 5 microm, 15 cm x 4.6 mm), isocratic elution with 10 mM 1-decane sulfonic acid sodium salt containing 10 mM sodium phosphate monobasic (pH 3.0)-ACN (60:40, v/v) and ultraviolet (UV) detection at 235 nm.     

Development and validation of a specific and sensitive GC-FID method for the determination of impurities in 5-chlorovaleroyl chloride

5-Chlorovaleroyl chloride (5-CVC) is commonly used as an alkylating agent in the synthesis of pharmaceutical intermediates, active ingredients, as well as other specialty chemicals. It is critical to monitor the impurities present in 5-CVC as they may have a direct impact on the impurity profile and quality of the final product. This paper describes the development and validation of a GC-FID method for the analysis of low level impurities of 5-CVC. This is the first method reported in the literature for the impurity determination of 5-CVC. The results of GC method development, with and without sample derivatization, are presented. The final method uses methanol for derivatization and separates methyl esters of 5-CVC and the key impurities, 4-pentenoyl chloride, 4-chlorovaleroyl chloride, 5-chlorohexanoyl chloride, and 4-methyl-5-chlorovaleroyl chloride. 3-Methoxypyridine was used in the sample solvent to enable the detection of 5-chlorovaleric acid (5-CVA) which is the major degradant of 5-CVC. The method was validated for specificity, linearity, accuracy, precision, sensitivity, and robustness. This simple and robust GC approach may be applicable to impurity analysis of other acid chlorides or acid halides.     

Method development and validation for the high-performance liquid chromatography assay of gastrodin in water extracts from different sources of Gastrodia elata Blume

Gastrodia elata Blume is commonly used as a medical herb in China for ameliorating headaches, dizziness, and convulsions. In previous studies, water extracts of G. elata Bl. (WGE) have demonstrated potential to act as therapeutic agents to improve depression-like symptoms in rats. As gastrodin (GAS) is a major active compound in WGE, its quantitation in WGE is important for quality control. The objective of this study was to develop an optimized and validated reversed-phase high-performance liquid chromatography method for the analysis of GAS in different sources of WGE. We evaluated the GAS content in varieties of G. elata Bl. including G. elata Bl. f. glauca S. Chow and G. elata Bl. f. elata. We also evaluated the GAS content of the latter variety from two different origins, Yun-nan and Hu-nan. The results indicate that the amount of GAS analyzed in WGE from G. elata Bl. f. glauca S. Chow is five times higher than that of G. elata Bl. f. elata from Yun-nan and Hu-nan. A significant difference in GAS content was observed between varieties of G. elata Bl., although not between locations of origin.     

Method development and validation for the analysis of meloxicam in tablets by CZE

A capillary zone electrophoresis assay for the analysis of meloxicam has been developed and validated. The influence of buffer concentration, buffer pH, methanol as organic modifier, capillary temperature, applied voltage and injection time was systemically investigated in a fused silica capillary (i.d. 50 microm, total length 44 cm and effective length 35.5 cm). Optimum results were obtained with a 100 mM borate buffer (pH 8.5) containing 5% methanol, capillary temperature 25 degrees C, applied voltage 20 kV and injection time 3 s hydrodynamic injection. The detection wavelength was set to 205 nm. Diflunisal was used as internal standard. The method showed good selectivity, accuracy, precision, linearity and sensitivity according to the evaluation of the validation parameters. The method was applied to the determination of six pharmaceutical preparations including two dosage forms. The relative standard deviation of 7 replicate analyses for each sample was less than 0.66%. The results were compared with a spectrophotometric method reported in literature and no significant difference was found statistically.     

H NMR-based metabonomic investigation of tributyl phosphate exposure in rats

Tributyl phosphate (TBP) is a toxic organophosphorous compound widely used in many industrial applications, including significant usage in nuclear processing. The industrial application of this chemical is responsible for occupational exposure and environmental pollution. In this study, ¹H NMR-based metabonomics has been applied to investigate the metabolic response to TBP exposure. Male Sprague-Dawley rats were given a TBP-dose of 15 mg/kg body weight, followed by 24 h urine collection, as was previously demonstrated for finding most of the intermediates of TBP. High-resolution ¹H NMR spectroscopy of urine samples in conjunction with statistical pattern recognition and compound identification allowed for the metabolic changes associated with TBP treatment to be identified. Discerning NMR spectral regions corresponding to three TBP metabolites, dibutyl phosphate (DBP), N-acetyl-(S-3-hydroxybutyl)-l-cysteine and N-acetyl-(S-3-oxobutyl)-l-cysteine, were identified in TBP-treated rats. In addition, the ¹H NMR spectra revealed TBP-induced variations of endogenous urinary metabolites including benzoate, urea, and trigonelline along with metabolites involved in the Krebs cycle including citrate, cis-aconitate, trans-aconitate, 2-oxoglutarate, succinate, and fumarate. These findings indicate that TBP induces a disturbance to the Krebs cycle energy metabolism and provides a biomarker signature of TBP exposure. We show that three metabolites of TBP, dibutylphosphate, N-acetyl-(S-3-hydroxybutyl)-l-cysteine and N-acetyl-(S-3-oxobutyl)-l-cysteine, which are not present in the control groups, are the most important factors in separating the TBP and control groups (p < 0.0023), while the endogenous compounds 2-oxoglutarate, benzoate, fumarate, trigonelline, and cis-aconetate were also important (p < 0.01).     

Development and validation of a new assay for assessing clot integrity

IntroductionResearch and routine laboratory assessment of clot integrity can be time consuming, expensive, and cannot be batched as it is generally performed in real time. To address these issues, we developed and validated a micro-titre based assay to quantify thrombogenesis and fibrinolysis, the purpose being to assess patients at risk of cardiovascular events by virtue of hypercoagulability. In further validation, thrombogenesis results were compared to similar indices from the thrombelastograph (TEG).MethodsOur assay determines three indices of thrombogenesis (lag time to the start of thrombus formation (LT), rate of clot formation (RCF), and maximum clot density (MCD)) and two of fibrinolysis (rate of clot dissolution (RCD) and time for 50% of the clot to lyse (T50)). Plasma was tested fresh and again after being frozen at − 70 °C. Some samples were tested immediately, others after being left at room temperature for up to 24 h.ResultsThe intra-assay coefficients of variation (CVs) of the three thrombogenesis measures (LT, RCF, MCD) and two fibrinolysis measures (RCD, T50) varied between 2.7 and 12.0% in fresh plasma and between 1.3% and 10.8% in frozen plasma respectively. Similarly, the inter-assay coefficients of variation of the thrombogenesis and fibrinolysis measures were 4.9–10.8% in fresh plasma and 2.2–6.5% in frozen plasma respectively. TEG assays intra- and inter assay CVs were around 25%. There were no significant differences in all plate assay indices up to 6 h at room temperature. Certain plate assay thrombogenesis data were comparable to TEG indices after analysis by Pearson's correlation. The reagent processing cost per sample is £15 for TEG and £2 for the plate assays.ConclusionOur micro-titre based assay assessing plasma thrombogenesis and fibrinolysis has good intra- and inter-assay CVs, can assess plasma up to 6 h after venepuncture, is more efficient (in terms of throughput) and is more economical than that of the TEG.     

磷酸可待因片含量测定方法的改进

磷酸可待因片(Codeine phosphate Tablets)是镇痛药和镇咳药,临床应用于中度疼痛.作为中枢性镇咳药尤其适用于无痰的干咳.为更好地控制其质量,针对其含量测定结果偏低问题,对<中国药典>2000年版二部该制剂的含量测定方法进行了适当改进,结果良好.     

毛细管气相色谱法测定糜蛋白酶中磷酸三丁酯残留量

目的建立用于检测糜蛋白酶原料药中磷酸三丁酯残留量的毛细管气相色谱法。方法供试品采用正己烷提取,内标法测定。TM—FFAP毛细管柱,载气为高纯氮气,正己烷为溶解介质,柱温为恒温160℃,进样口温度为200℃,检测器温度为230℃,进样量1．0μL,分流进样,分流比1：5,FID检测器。结果被测物磷酸三丁酯在所考察的浓度范围内呈现良好线性关系,r=0．9966,平均回收率为96．28％～103．54％,精密度的RSD〈5％,完全符合残留溶剂测定的技术要求。结论本试验建立的方法简单、准确、精密度高,可用于糜蛋白酶中磷酸三丁酯残留量的测定。     

Method development and validation of capillary sodium dodecyl sulfate gel electrophoresis for the characterization of a monoclonal antibody

A capillary sodium dodecyl sulfate gel electrophoresis (cSDS) method has been developed and qualified for purity and impurity analysis of monoclonal antibodies. This method was optimized and qualified for the analysis of monoclonal antibody (mAb1) under reduced and non-reduced conditions.     

Development and validation of a quantitative assay for raloxifene by capillary electrophoresis

The migration behavior of raloxifene was investigated by capillary electrophoresis (CE). The influence of different parameters (nature and concentration of the running buffer, pH and applied voltage) on migration time, peak symmetry and efficiency was systematically investigated. A buffer consisting of 20 mM acetate buffer of pH 4.5 was found to provide a very efficient and stable electrophoretic system for the analysis of raloxifene. The optimized method was validated with respect to precision, linearity, limits of detection and quantification, accuracy and robustness. The applicability of the assay was demonstrated by analyzing this drug in human plasma and pharmaceutical preparations.     

Immunoassay method validation for a modified EMIT phencyclidine assay.

Immunoassay drug testing methods, that have been modified from the manufacturers' recommended procedure to be used for the analysis of federally regulated specimens or other forensic samples require evaluation to ensure their scientific validity. These validation studies must demonstrate the accuracy, precision, and linearity of the modified immunoassay around the cutoff concentration, substantiate adequate rate separation, and verify the ability of the assay to differentiate positive and negative samples. Modification of the EMIT d.a.u. phencyclidine assay in order to achieve the federally mandated cutoff concentration of 25 ng/mL is common. This study describes the validation of a modified EMIT phencyclidine assay and a protocol that allows for the evaluation of similarly modified immunoassays.     

Choosing the calibration model in assay validation

Data transformations and weighting schemes are normally used to obtain the best-fit of standard curves in bioanalysis and the calibration model is usually selected during prevalidation. In the present study, a comparison has been made between unweighted and weighted (1/x, 1/x2, and 1/square root of x) regression models with or without an intercept in achieving the best-fit for the standard curve of CDRI compound 81/470, a new anthelmintic agent, in cow milk. Validation samples in milk at the LLOQ, medium, and high concentrations were also analysed by each of the calibration models. An unweighted regression equation with an intercept overestimated the concentrations at the LLOQ. An unweighted equation without intercept and weighted equations with or without an intercept significantly minimized the bias at the LLOQ without distorting the results at higher concentrations. Hence, an unweighted equation for a straight line passing through the origin was found to be the best model for a standard curve of 81/470 in milk. Similar results were obtained for 81/470 and UMF-078 in serum and plasma, respectively. Bioanalysts should routinely test these models to obtain the best fit model for their calibration curves as part of their assay validation not during prevalidation.     

Development and validation of a high-performance liquid chromatography assay and a capillary electrophoresis assay for the analysis of adenosine and the degradation product adenine in infusions

A high-performance liquid chromatography (HPLC) method and a capillary electrophoresis (CE) method for the analysis of adenosine and the degradation product adenine in infusion solutions have been developed and validated. The HPLC separation of the analytes was achieved on a RP-18 column, using a mobile phase, consisting of 20mM ammonium acetate, pH 6.0, containing 5% of acetonitrile at a flow rate of 1ml/min. Thymidine was used as internal standard. The CE separation was performed in a fused-silica capillary with a 100mM sodium phosphate buffer, pH 2.7, at an applied voltage of 25kV, using cytidine as internal standard. The assays were validated with regard to linearity, range, limit of detection (LOD), limit of quantitation (LOQ), specificity, and precision. Both methods were specific allowing reliable quantification of the analytes. Compared to the CE method, HPLC analysis yielded a two- to five-fold lower LOD. With respect to analysis time, CE was faster than HPLC. The applicability of both methods for the determination of the purity and stability of adenosine in the infusion solutions is demonstrated.     

Development and validation of a cell-based assay for the nuclear receptor retinoid-related orphan receptor gamma

The nuclear receptor retinoid-related orphan receptor gamma (RORγ) has become an attractive target for drug discovery due to its important role in the development and differentiation of Th17 cells, a subset of T cells that produce interleukin-17 and are involved in the pathogenesis of human inflammatory and autoimmune diseases. To facilitate the drug discovery efforts in this area, we have developed a cellular assay for screening for RORγ inverse agonists. We stably engineered a tetracycline-inducible Gal4 DNA-binding domain/RORγ ligand-binding domain fusion protein into an upstream activation sequence driven-beta-lactamase reporter gene cell line. Due to its constitutive activity, the induced Gal4-RORγ expression leads to increased reporter activity, which can be knocked down using RORγ ligand-binding domain-specific RNA interference oligos. Using this assay, we tested several recently reported ligands for RORγ and observed varying levels of partial inverse agonist activity at μM concentrations. Additionally, we screened a small library of biologically active compounds with this assay and demonstrated its robustness and usefulness in high-throughput screening and follow-up studies for this emerging drug target.     

Development and validation of a stability-indicating high performance liquid chromatographic assay for benoxinate

Benoxinate is a local anaesthetic used for ophthalmic applications. The aim of this study was to develop a rapid and simple stability-indicating method for the determination of benoxinate formulated for ophthalmic use, evaluate its long-term stability and identify its major degradation product. Benoxinate was eluted on a 10 microm Spherisorb phenyl column, 250 x 3.2 mm, with a mobile phase consisting of acetonitrile-buffer (pH 3.5) (35:65, v/v), pumped at 0.8 ml min(-1) flow rate. The buffer was composed of sodium dihydrogen phosphate (50 mM), sodium hydrogen sulfate (2.5 mM) and 1-heptanesulfonic acid sodium salt (5 mM). The analyte was quantified spectrophotometrically at 308 nm. The chromatograms of benoxinate formulations obtained by this method showed benoxinate (t = 4.5 min) well resolved from its degradation product (t = 2.3 min), which was separately identified by means of HPLC-MS as 4-amino-3-butoxybenzoic acid. The assay was demonstrated to have high accuracy, precision and linearity. The method was implemented in investigating the long-term stability of benoxinate 0.4% ophthalmic solutions. The method was found to be simple, quick and selective in determining benoxinate concentrations in fresh and aged preparations.     

Development and validation of a radioreceptor assay for the determination of morphine and its active metabolites in serum

This article describes the development and validation of a radioreceptor assay for the determination of morphine and morphine-6-β-glucuronide (M6G) in serum. The assay is based on competitive inhibition of the μ-opioid-selective radiolabeled ligand [³H]-DAMGO by opioid ligands (e.g. M6G) for binding to the striatal opioid receptor. The assay has been validated according to the Washington Conference Report on Analytical Method Validation. The radioreceptor assay can be performed in serum without prior pre-treatment of the sample. Direct addition of the sample results in no significant loss in maximal binding sites, and therefore, no loss in sensitivity. The assay proves to be selective for a multitude of opioid agonists and antagonists (e.g. morphine IC₅₀ = 4.1 nM and M6G IC₅₀ = 12.8 nM). Moreover, morphine-3-glucuronide (M3G) displays a low affinity (IC₅₀ = 1100 nM) for the μ-opioid receptor and according to the literature demonstrates no analgesic activity. This makes discrimination, in relation to the analgesic effect, of the two metabolites of morphine possible. The assay is fast (assay time <4 h, analysis 5 min/sample), easy and the sensitivity (limit of detection (LOD) = 1.6 nM M6G-equivalents) is such that very potent agonists, like morphine and M6G, can be measured at the desired serum levels. The assay is accurate (<18%), but precision is limited if measured over several days (>35%). The assay is most accurate and precise if measured over a range from 3.5 to 40 nM M6G-equivalents. Based on the limited inter-assay precision, we propose to use this receptor assay mainly as a screening tool for neonates treated with morphine.     

Development and validation of a rapid HPLC method for the determination of oseltamivir phosphate in Tamiflu and generic versions

Oseltamivir phosphate (OP) is an antiviral drug that is used in the treatment and prophylaxis of both influenza A and influenza B. It is effective against all known influenza viruses than can infect humans, including pandemic influenza viruses and may be the most appropriate antiviral option against avian influenza caused by H5N1 virus. Tamiflu, the registered trademark used under exclusive license by Roche laboratories with OP as active pharmaceutical ingredient, is considered the best treatment for the bird flu disease. A simple, selective, linear, accurate and precise HPLC method was developed and validated for rapid assay of OP aimed to the quality control of Tamiflu capsules and generic versions. Isocratic elution at a flow rate of 1.2 mL/min was employed on a Zorbax CN column (150 mm x 4.6mm; 5 microm) at ambient temperature. The mobile phase consisted of methanol and 0.04 M formic acid pH 3.0 (50:50, v/v). The UV detection wavelength was 226 nm and 20 microL of sample was injected. Sotalol hydrochloride was used as the internal standard (IS). The retention times for OP and IS were 3.40 and 2.25 min, respectively. The method was successfully applied to commercial pharmaceuticals, Tamiflu and generic versions. The proposed method could be applicable for routine analysis of OP and monitoring of the quality of marketed drugs as possibly counterfeit Tamiflu.