UV spectrophotometric determination of bioflavonoids from a semisolid pharmaceutical dosage form containing Trichilia catigua Adr. Juss and Ptychopetalum olacoides Bentham standardized extract: analytical method validation and statistical procedures

A precise, accurate, and sensitive UV spectrophotometric method was developed and validated for routine quantification of total bioflavonoids, expressed as rutin, from a topical oil-in-water pharmaceutical emulsion containing the extract of Trichilia catigua Adr. Juss and Ptychopetalum olacoides Bentham. The method was validated experimentally, and the data were treated rigorously by statistical analysis. The following analytical parameters were assessed: linearity, specificity, intra- and interrun precision measured as relative standard deviation (RSD, %), intra- and interrun accuracy (E, %), recovery (Rec., %), limit of detection (LOD, microg/mL), and limit of quantification (LOQ, microg/mL). The UV spectrophotometric method was linear (r = 0.9995) for standard rutin over the concentration range of 5.0-15.0 microg/mL with specificity for total bioflavonoids (expressed as rutin) at 361.0 nm with an absence of interferents from the complex matrix; RSD of < or = 1.79%, intrarun (E = 97.88 +/- 1.75 to 99.0 +/- 0.33%) and interrun (E = 98.38 +/- 1.12 to 100.79 +/- 1.30%) accuracy; Rec. = 98.64 +/- 0.42 to 100.74 +/- 0.41%; LOD = 0.20 microg/mL; and LOQ = 0.30 microg/mL.     

Rapid method for determination of chlormequat residues in tomato products by ion-exchange liquid chromatography/electrospray tandem mass spectrometry

A rapid method has been devised for the direct determination of chlormequat in tomato samples. No clean-up is required, and analysis uses ion-exchange liquid chromatography/tandem mass spectrometry interfaced with electrospray ionization (LC/ESI-MS/MS). A cation-exchange column was used with an aqueous ammonium acetate/acetonitrile mixture as the mobile phase under isocratic conditions. The method was validated in terms of detection limits (LOD), quantitation limits (LOQ), linearity, recovery, precision and accuracy. Good results in the low micro g kg(-1) level were obtained for the LOD and LOQ of chlormequat in tomato samples. Comparison of solvent and matrix-matched calibration curves demonstrated the absence of significant matrix effects and the feasibility of using external calibration. Linearity was established over two orders of magnitude by performing homoscedasticity and Mandel fitting statistical tests. The absence of both constant and proportional systematic errors was verified by evaluating the recovery function, demonstrating good method accuracy. Excellent precision in terms of intra-day repeatability was calculated (RSD% <3.4). Extraction recoveries from tomato products were calculated, by using a labelled internal standard (d(4)-chlormequat), to be in the 93 +/- 5-99 +/- 7% range. The applicability of the method to the determination of chlormequat residues in tomato products was demonstrated.     

Validation assay for total flavonoids, as rutin equivalents, from Trichilia catigua Adr. Juss (Meliaceae) and Ptychopetalum olacoides Bentham (Olacaceae) commercial extract

An ultraviolet spectrophotometric method was validated for total flavonoid quantitation, as rutin equivalents, present in the Trichilia catigua Adr. Juss (Meliaceae) and Ptychopetalum olacoides Bentham (Olacaceae) commercial extract. Parameters as linearity, interval (range), specificity, estimated limit of detection (LOD, microg/mL), estimated limit of quantitation (LOQ, microg/mL), recovery (R, %), precision or relative standard deviation (RSD, %), and accuracy (E, %) were established. The analytical method was validated according to the experimental results: correlation coefficient (r = 0.9997); interval (RSD = 0.15-0.47%; E = 98.98-101.24%); specificity to total flavonoids quantitation, as rutin equivalents, at wavelength 361.0 nm; LOD = 0.09 microg/mL and LOQ = 0.27 microg/mL; R = 99.36-102.14%; adequate intra- and interrun precision (0.30-0.49% and 0.31-0.81%), and intra- and interrun accuracy (100.60-102.38% and 98.58-100.38%).     

Determination of carbamazepine and its impurities iminostilbene and iminodibenzyl in solid dosage form by column high-performance liquid chromatography

An accurate and precise RP-HPLC method was developed and validated for the determination of carbamazepine and its impurities iminostilbene and iminodibenzyl in a tablet formulation with fluphenazine as an internal standard. Buffer-methanol (50 + 50, v/v) was used as the mobile phase. During validation, specificity, linearity, precision, accuracy, LOD, LOQ, and robustness of the method were tested. The method was proven to be specific against placebo interference. Linearity was evaluated over the concentration range of 100-500, 0.05-0.25, and 0.1-0.5 microg/mL, and the r values were 0.9994, 0.9997, and 0.9979 for carbamazepine, iminostilbene, and iminodibenzyl, respectively. Intraday precision of the method was good, and RSD was below 2% for all analytes. The accuracy of the method ranged from 100.69 to 102.10, 99.76 to 102.66, and 99.26 to 100.08% for carbamazepine, iminostilbene, and iminodibenzyl, respectively. LOD was 0.0125, 0.025, and 0.05 microg/mL and LOQ was 0.05, 0.05, and 0.1 microg/mL for carbamazepine, iminostilbene, and iminodibenzyl, respectiviely. Robustness of the method was proven by using a chemometric approach. The method was successfully applied to the analysis of commercially available carbamazepine tablets and showed good repeatability, with RSD below 2%.     

Development and validation of a new HPLC-UV method for the simultaneous determination of triclabendazole and ivermectin B1a in a pharmaceutical formulation

A rapid, simple, and sensitive RP-HPLC analytical method was developed for the simultaneous determination of triclabendazole and ivermectin in combination using a C18 RP column. The mobile phase was acetonitrile-methanol-water-acetic acid (56 + 36 + 7.5 + 0.5, v/v/v/v) at a pH of 4.35 and flow rate of 1.0 mL/min. A 245 nm UV detection wavelength was used. Complete validation, including linearity, accuracy, recovery, LOD, LOQ, precision, robustness, stability, and peak purity, was performed. The calibration curve was linear over the range 50.09-150.26 microg/mL for triclabendazole with r = 0.9999 and 27.01-81.02 microg/mL for ivermectin with r = 0.9999. Calculated LOD and LOQ for triclabendazole were 0.03 and 0.08 microg/mL, respectively, and for ivermectin 0.07 and 0.20 microg/mL, respectively. The intraday precision obtained was 98.71% with RSD of 0.87% for triclabendazole and 100.79% with RSD 0.73% for ivermectin. The interday precision obtained was 99.51% with RSD of 0.35% for triclabendazole and 100.55% with RSD of 0.59% for ivermectin. Robustness was also studied, and there was no significant variation of the system suitability of the analytical method with small changes in experimental parameters.     

Determination of dodine in fruit samples by liquid chromatography electrospray ionization-tandem mass spectrometry

A rapid and sensitive LC/electrospray ionization-MS/MS method has been developed for the determination of dodine in fruit samples. Based on a liquid-liquid extraction of 10 g solid fruit homogenate using an acetone-dichloromethane-hexane mixture and acetate ammonium buffer (pH 4.5), this LC/MS/MS procedure was characterized by recoveries above 50%, with good intra-assay precision (RSD < 13%) and interassay precision (RSD < 18%) for seven different matrixes (apple, apricot, cherry, peach, pear, plum, and quince). This method was validated from 5 to 500 microg/kg according to standard guidelines. Its LOD (1 microg/kg) and LOQ (5 microg/kg) were in accordance with recommendations of the European legislation defined for infant food [maximum residue level (MRL) = 10 microg/kg]. The whole procedure was finally tested on 1022 fruit samples intended for commercialization, both infant food samples and samples not intended in particular for babies. In this study, dodine was detected in 27 samples; none exhibited a concentration higher than the MRL.     

A comparative study of first-derivative spectrophotometry and column high-performance liquid chromatography applied to the determination of repaglinide in tablets and for dissolution testing

A fast and reliable method for the determination of repaglinide is highly desirable to support formulation screening and quality control. A first-derivative UV spectroscopic method was developed for the determination of repaglinide in tablet dosage form and for dissolution testing. First-derivative UV absorbance was measured at 253 nm. The developed method was validated for linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ) in comparison to the U.S. Pharmacopeia (USP) column high-performance liquid chromatographic (HPLC) method. The first-derivative UV spectrophotometric method showed excellent linearity [correlation coefficient (r) = 0.9999] in the concentration range of 1-35 microg/mL and precision (relative standard deviation < 1.5%). The LOD and LOQ were 0.23 and 0.72 microg/mL, respectively, and good recoveries were achieved (98-101.8%). Statistical comparison of results of the first-derivative UV spectrophotometric and the USP HPLC methods using the t-test showed that there was no significant difference between the 2 methods. Additionally, the method was successfully used for the dissolution test of repaglinide and was found to be reliable, simple, fast, and inexpensive.     

Determination of total iodine in foods and dietary supplements using inductively coupled plasma-mass spectrometry

A method was developed and validated for the determination of total iodine in a wide variety of food products and dietary supplements. The method involves a unique sample digestion with a KOH solution in an oven or by using an open-vessel microwave system. After digestion, a stabilizer is added and the solution is taken to volume, then filtered and analyzed either directly or after dilution. The amount of iodine is determined with inductively coupled plasma-mass spectrometry (ICP-MS). The method was validated by experiments to determine its precision, accuracy, linearity, specificity, ruggedness, and robustness. The LOQ of this method is 25-50 microg/kg. The method demonstrated an average RSD of 2.27% during analysis of milk powder and 4.30% during analysis of a dietary supplement tablet reference material. The accuracy of the method as determined with these same reference materials was 100 and 94.2%, respectively. The method has been used successfully on commodity foods, processed foods, dairy products, pet food, infant formula, animal feed, mineral premixes, and a variety of dietary supplements.     

Enantioselective separation and determination of the dinotefuran enantiomers in rice,tomato and apple by HPLC

An effective chiral analytical method was developed for the resolution and determination of dinotefuran enantiomers in rice, tomato and apple samples. Dinotefuran enantiomers were baseline‐separated and determined on a novel chiral column, ChromegaChiral CCA, with n‐hexane–ethanol–methanol (85:5:10, v/v/v) as the mobile phase at a flow rate of 1.0 mL/min with UV detection at 270 nm. The resolution of dinotefuran enantiomers was about 1.8. The first eluted enantiomer was (+)‐dinotefuran and the second eluted one was (?)‐dinotefuran. The effects of mobile‐phase composition and column temperature on the enantioseparation were evaluated. The method was validated for linearity, repeatability, accuracy, LOD and LOQ. LOD was 0.15 mg/kg in rice and tomato, 0.05 mg/kg in apple, with an LOQ of 0.5 mg/kg in rice and tomato, 0.2 mg/kg in apple. The average recoveries of the pesticide from all matrices ranged from 75.8 to 92.9% for all fortification levels The precision values associated with the analytical method, expressed as RSD values, were <16.5% for the pesticide in all matrices. The methodology was successfully applied for the enantioselective analysis of dinotefuran enantiomers in real samples, indicating its efficiency in investigating the environmental stereochemistry of dinotefuran in food matrix.     

Confirmatory method for the determination of volatile congeners and methanol in Turkish raki according to European Union Regulation (EEC) No. 2000R2870: single-laboratory validation

A method described by European Union Regulation (EEC) No. 2000R2870 was validated and supported by GC/MS analysis for the determination of volatile congeners and methanol in Turkish raki. The method was validated in terms of specificity, accuracy, precision, LOD, LOQ, linearity, and robustness. The specificity of the method was demonstrated, and the method showed excellent accuracy (97.5-100.1%). Linearity was checked in the ranges of 0.200-26.390 mg/100 mL for more volatile compounds and 1.155-48.00 mg/100 mL for less volatile compounds, after concentrations found in Turkish raki were taken into account. The calibration curves of all analytes showed good linearity (R2 > 0.998). The within- and between-day precision (RSD) values of 11 analytes were in the range of 0.18-4.50%. The LOD and LOQ values were in the range of 0.014-0.362 and 0.045-1.085 mg/100 mL, respectively. The method can be used as an absolute quantification method for the determination of volatile congeners and methanol in Turkish raki and for QC.     

Chiral determination of a novel acaricide cyflumetofen enantiomers in cucumber,tomato, and apple by HPLC

A simple chiral analytical method was developed for the enantiomeric determination of cyflumetofen in cucumber, tomato, and apple by normal‐phase HPLC. The effects of mobile phase composition and column temperature on the enantioseparation were evaluated. Excellent separation was achieved at 25°C on a Chiralpak AD‐H column, with a mixture of n‐hexane and 2‐propanol (95:5, v/v) as mobile phase at a flow rate of 1.0 mL/min detecting at 234 nm. The resolution of cyflumetofen enantiomers was up to 5.5. The elution order of the enantiomers was determined by an online OR‐2090 detector, which was performed under the same chromatographic conditions. The first eluted enantiomer was (–)‐cyflumetofen and the second eluted one was (+)‐cyflumetofen. The method was validated for linearity, repeatability, accuracy, LOD, and LOQ. LOD ranged from 0.1 to 0.15 mg/kg, with the LOD varying from 0.33 to 0.5 mg/kg for each enantiomer, respectively. The average recoveries of the pesticide ranged from 71.4 to 102.0% at all fortification levels. The precision values associated with the analytical method, expressed as RSD values, were below 14.8% in all matrices. The method was then successfully applied to detect cyflumetofen enantiomers in real samples.     

Development of a simple and sensitive high-performance liquid chromatography method for determination of glucosamine in pharmaceutical formulations

A column high-performance liquid chromatography (HPLC) method was developed for the determination of glucosamine in dosage forms. Glucosamine was derivatized by addition of a solution containing orthophthaldialdehyde. The HPLC separation was achieved on a Spherimage 80 ODS2 column (250 x 4 mm id, 5 microm particle size) using an isocratic mobile phase containing phosphate buffer-methanol (90 + 10, v/v, pH 6.50) and methanol-tetrahydrofuran (97 + 3, v/v) in proportions of 85 + 15 at a flow rate of 1 mL/min, followed by fluorescence detection. The method was validated for specificity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). The detector response for glucosamine HCI was linear over the concentration range of 0.1-20 microg/mL with a correlation coefficient of 0.9980. The accuracy was between 99.4 and 100.8%. The LOD and the LOQ were 0.009 and 0.027 microg/mL, respectively. The method was applied to determination of glucosamine in solid dosage forms.     

Development and validation of a HPLC method for 4,7-phenanthroline-5,6-dione I and identification of its major impurity by HPLC-MS-APCI

In this study, the development and validation of an analytical method for the assay of 4,7-phenanthroline-5,6-dione I (dione I) using high-performance liquid chromatography (HPLC) and the determination of its synthetic impurities by employing the method in HPLC-mass spectrometry with atmospheric-pressure chemical ionization and photodiode-array UV detection is reported. The results show that dione I is eluted as a spectrally pure peak resolved from its impurities. 5-Bromo,4-7-phenanthroline is identified as the main impurity. This is supported by elemental analysis of the dione I, which demonstrated the presence of bromine. Validation parameters such as specificity and selectivity, linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), ruggedness, stability, and system suitability, which are evaluated for this method. The LOD and LOQ are 2.0 microg/mL and 50 microg/mL with a 0.50% relative standard deviation (%RSD), respectively. The calibration curves showed good linearity over the concentration range of 0.05-1.50 mg/mL. The correlation coefficient is > 0.9991 in each case. The %RSD values for intra- and interday precision studies are < 0.40%.     

Determination of fenthion and oxidation products in personal protection equipment by gas chromatography

A method for the co-extraction and simultaneous chromatographic determination of fenthion and its five oxidation products (or metabolites) fenoxon, fenoxon-sulfoxide, fenoxon-sulfone, fenthion-sulfoxide, and fenthion-sulfone in personal protection equipment (PPE) of pesticide applicators was developed and validated. Capillary gas chromatography-nitrogen-phosphorus detection was used for the analytical determination of all the aforementioned compounds over the concentration range of 0.1-0.5 microg/mL. All necessary validation criteria of the method were met. The method was found to be highly selective, accurate, and precise, gave satisfactory recovery (>70%) and RSD values (<20%) for fenthion and its metabolites in all tested specimens of personal protection equipment and in air samplers. The limit of quantification (LOQ) and linearity were determined for the most relevant PPE (i.e. inner coverall) for the parent compound and for the metabolites. The LOQ values ranged from 0.05 to 0.1 ppb while the linearity in the tested range of 0.1-0.5 ppm had r(2)>0.994 for all analytes.     

Method validation for the analysis of 169 pesticides in soya grain, without clean up, by liquid chromatography-tandem mass spectrometry using positive and negative electrospray ionization

Part of a comprehensive study on the comparison of different extraction methods, GC-MS(/MS) and LC-MS/MS detection methods and modes, for the analysis of soya samples is described in this paper. The validation of an acetone-based extraction method for analysis of 169 pesticides in soya, using LC-MS/MS positive and negative electrospray ionisation (ESI) mode, is reported. Samples (5 g) were soaked with 10 g water and subsequently extracted with 100 mL of a mixture of acetone, dichloromethane and light petroleum (1:1:1), in the presence of 15 g anhydrous sodium sulphate. After centrifugation, aliquots of the extract were evaporated and reconstituted in 1.0 mL of methanol, before direct injection of the final extract (corresponding with 0.05 g soya mL(-1)) into the LC-MS/MS system. Linearity, r(2) of calibration curves, instrument limit of detection/quantitation (LOD/LOQ) and matrix effect were evaluated, based on seven concentrations measured in 6-fold. Good linearity (at least r(2)> or =0.99) of the calibration curves was obtained over the range from 0.1 or 0.25 to 10.0 ng mL(-1), corresponding with pesticide concentrations in soya bean extract of 2 or 5-200 microg kg(-1). Instrument LOD values generally were 0.1 or 0.25 ng mL(-1). Matrix effects were negligible for approximately 90% of the pesticides. The accuracy, precision and method LOQ were determined via recovery experiments, spiking soya at 10, 50, 100 microg kg(-1), six replicates per level. In both ESI modes, method LOQ values were mostly 10 or 50 microg kg(-1) and more than 70% of pesticides analysed by each mode met the acceptability criteria of recovery (70-120%) and RSD (< or =20%), at one or more of the three levels studied. A fast, easy and efficient method with acceptable performance was achieved for a difficult matrix as soya, without cleanup.     

Development and in-house validation of a liquid chromatography-electrospray-tandem mass spectrometry method for the simultaneous determination of Sudan I, Sudan II, Sudan III and Sudan IV in hot chilli products

An accurate method based on the use of reversed-phase (RP) liquid chromatography-tandem mass spectrometry interfaced with electrospray (LC-ESI-MS/MS) was devised for the determination of Sudan I, Sudan II, Sudan III and Sudan IV in hot chilli food samples. A simple sample treatment procedure entailing the use of an extraction step with acetone without clean-up was developed. A C18 column with an aqueous formic acid/methanol mixture as the mobile phase was used under isocratic conditions. Mass spectral acquisition was done in positive ion mode by applying selected reaction monitoring of three fragmentation transitions per compound to provide a high degree of selectivity. The method was in-house validated in terms of detection limits (LOD), quantitation limits (LOQ), linearity, sensitivity, accuracy, recovery, and selectivity on two kinds of hot chilli sauces. Good results in the low ng/g level were obtained for LOD and LOQ of all analytes in matrices. Under both intra-day repeatability (R.S.D. between 1 and 13%) and intermediate precision (about 5-15% R.S.D. for both chilli sauce matrices) conditions, precision proved to be typical of determinations based on electrospray LC-MS and acceptable for routine monitoring purposes. Extraction recoveries for all four azo-dyes in chilli tomato sauce ranged from 92 to 103% at a spiking level of 5 microg/kg, whereas values between 72 and 97% were calculated in chilli tomato and cheese sauce at the same concentration level. The applicability of the method to the determination of Sudan azo-dyes in hot chilli products was demonstrated.     

Development and validation of a solid-phase extraction and gas chromatography-tandem mass spectrometry method for the determination of isopropyl-9H-thioxanthen-9-one in carton packaged milk

A simple and efficient method for the determination of isopropyl-9H-thioxanten-9-one (ITX) in different fat content milk samples and baby milk samples stored in packaged cartons was developed and validated. Samples were extracted using solid-phase extraction (SPE) and analysed by gas chromatography-tandem mass spectrometry operated in selected reaction monitoring mode (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values in the low microg/L were achieved, whereas linearity was established within 0.5-500 microg/L range. Good precision was obtained both in terms of intra-day repeatability and inter-day precision on two concentration levels (RSD% lower than 2%). Good percentage recoveries were obtained (92.0-102.0%) even in the presence of high amount of fat. Finally, the developed method was successfully applied to analyse a number of commercial milk samples with different fat content and baby milk samples.     

Octadecyl-bonded silica membrane disk modified with Cyanex302 for pre-concentration and determination of iron in food products

A simple and rapid method using an octadecyl-bonded silica membrane disk impregnated with Cyanex302 is described for the pre-concentration and determination of iron. The influence of various parameters on sorption and elution of Fe(III) were systematically investigated. The sorption of Fe(III) at pH 3.2 was quantitative (99.3 +/- 1.1%). It was completely recovered using 20 mL 5.0 M HCI and determined by flame atomic absorption spectrometry. Breakthrough volume of the modified disk for Fe(III) was >2000 mL, pre-concentration factor was >100, and reusability up to 28 cycles. The LOD and LOQ for Fe(III) were 0.45 microg/L and 1.51 microg/L, respectively, while precision for its determination in terms of RSD was < or =2.1%. This method was applied for Fe(III) determination in milk, fortified flour, cocoa powder, tea, and black pepper. To validate the procedure, EPA Method Standard (QC standard 21) was analyzed for Fe(III).     

Quantitative determination and validation of avermectin B1a in commercial products using quantitative nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance is defined as a quantitative spectroscopic tool that enables a precise determination of the number of substances in liquids as well as in solids. There is few report demonstrating the application of NMR in the quantification of avermectin B_1a (AVB_1a); here, a proton nuclear magnetic resonance spectroscopy (¹H NMR) using benzene [1‐methoxy‐4‐(2‐nitroethyl) (PMN)] as an internal standard and deuterochloroform as an NMR solvent was tested for the quantitative determination of AVB_1a. The integrated signal of AVB_1a at 5.56 ppm and the signal of PMN at 8.14 ppm in the ¹H NMR spectrum were used for quantification purposes. Parameters of specificity, linearity, accuracy, precision, intermediate precision, range, limit of detection (LOD), limit of quantification (LOQ), stability and robustness were validated. The established method was accurate and precise with good recovery (98.86%) and relative standard deviation (RSD) of assay (0.34%) within the linearity of the calibration curve ranging from 5.08 to 13.58 mg/ml (R² = 0.9999). The LOD and LOQ were 0.009 and 0.029 mg/ml, which indicated the excellent sensitivity of the method. The stability of the method was testified by a calculated RSD of 0.11%. The robustness was testified by modification of four different parameters, and the differences among each parameter were all less than 0.1%. Comparing with the assay described by the manufacturer of avermectin tablets, there was no significant difference between the assay obtained by HPLC and quantitative NMR (qNMR), which indicated qNMR was a simple and efficient method for the determination of AVB_1a in commercial formulation products. Copyright © 2014 John Wiley & Sons, Ltd.     

超高效液相色谱-大气压化学电离-串联质谱法测定烘焙咖啡中丙烯酰胺

建立了烘焙咖啡中丙烯酰胺的超高效液相色谱-大气压化学电离-串联质谱（UHPLC-APCI-MS/MS）分析方法。样品经甲醇提取，HLB固相萃取（SPE）小柱净化，Brownlee validated AQ C18色谱柱分离，采用大气压化学电离（APCI）源，正离子扫描和多反应监测（MRM）模式对丙烯酰胺进行检测，内标法定量。结果表明，丙烯酰胺在0.5~100.0 μg/L范围内具有良好的线性关系，相关系数（r²）为0.999，方法检出限为5.0 μg/kg，定量限为10.0 μg/kg。在100.0、200.0和1000.0 μg/kg添加水平下，丙烯酰胺的回收率为94.6%~115.0%，相对标准偏差（RSD）值为2.8%~3.6%（n=6）。本方法采用APCI源作为离子化方式，能有效地减少咖啡基质对丙烯酰胺的基质干扰，前处理简单，灵敏度高，适用于咖啡中丙烯酰胺的日常检测。