Simultaneous determination of polyphenols and triterpenes in pomegranate peel based on high‐performance liquid chromatography fingerprint by solvent extraction and ratio blending method in tandem with wavelength switching

Traditionally, pomegranate (Punica granatum L.) has been consumed as fresh fruit or as pomegranate juice. Pomegranate peel, the dried husk of P· granatum, is an important herbal medicine for treating diarrhea, hemostasis and insect‐induced abdominal pain in China. However, the quality control methods for pomegranate peel remain unsatisfactory. In this work, a new HPLC‐based qualitative and quantitative method for quality control of pomegranate peel was developed and validated for the simultaneous determination of polyphenols and triterpenes (including punicalagins A and B, ellagic acid, oleanolic acid and ursolic acid) by solvent extraction and ratio blending method in tandem with wavelength switching. The average recoveries were 98.07–100.61% with relative standard deviation no more than 4.27%. In addition, the fingerprint analysis was conducted to interpret the consistency of the quality test. Thirteen characteristic peaks were selected to evaluate the similarities of 16 batches of pomegranate peel. The similarities of samples were all more than 0.80, indicating that the samples from different areas of China were consistent. The results demonstrated that quantitative analysis and the HPLC fingerprint as a characteristic distinguishing method combining similarity evaluation can be successfully used to assess the quality and to identify the authenticity of pomegranate peel.     

Quality control and discrimination of pericarpium citri reticulatae and pericarpium citri reticulatae viride based on high-performance liquid chromatographic fingerprints and multivariate statistical analysis

High-performance liquid chromatographic (HPLC) fingerprints of Pericarpium Citri Reticulatae (PCR) and Pericarpium Citri Reticulatae Viride (PCRV) were firstly measured for deliberately collected 39 authentic samples and 21 commercial samples. Both correlation coefficients of similarity for chromatograms and absolute peak areas of characteristic compounds were calculated for quantitative expression of the HPLC fingerprints. After principal component analysis (PCA) successfully distinguished the ‘mixed peels’ samples from authentic samples, partial least squares-linear discrimination analysis (PLS-LDA) was then effectively applied to class separation between authentic PCR and PCRV. Furthermore, the unequivocally determined compounds, hesperidin, nobiletin and tangeretin, were screened out by loadings plots of PCA and PLS-LDA. The results indicated that they could be used as chemical markers for discrimination among different groups of samples. The proposed method shows an efficient strategy for quality control of PCR and PCRV, which cannot only distinguish the ‘mixed peels’ but also discriminate authentic PCR and PCRV. This method has potential perspective for quality control of traditional Chinese medicine (TCM).     

Fabrication of novel nanoporous array anodic alumina solid-phase microextraction fiber coating and its potential application for headspace sampling of biological volatile organic compounds

In the study, nanoporous array anodic alumina (NAAA) prepared by a simple, rapid and stable two-step anodic oxidization method was introduced as a novel solid-phase microextraction (SPME) fiber coating. The regular nanoporous array structure and chemical composition of NAAA SPME fiber coating was characterized and validated by scanning electron microscopy and energy dispersive spectroscopy, respectively. Compared with the commercial polydimethylsiloxane (PDMS) SPME fiber coating, NAAA SPME fiber coating achieved the higher enrichment capability (1.7–4.7 folds) for the mixed standards of volatile organic compounds (VOCs). The selectivity for volatile alcohols by NAAA SPME fiber coating demonstrated an increasing trend with the increasing polarity of alcohols caused by the gradually shortening carbon chains from 1-undecanol to 1-heptanol or the isomerization of carbon chains of some typical volatile alcohols including 2-ethyl hexanol, 1-octanol, 2-phenylethanol, 1-phenylethanol, 5-undecanol, 2-undecanol and 1-undecanol. Finally, NAAA SPME fiber coating was originally applied for the analysis of biological VOCs of Bailan flower, stinkbug and orange peel samples coupled with gas chromatography–mass spectrometry (GC–MS) detection. Thirty, twenty-seven and forty-four VOCs of Bailan flower, stinkbug and orange peel samples were sampled and identified, respectively. Moreover, the contents of trace 1-octanol and nonanal of real orange peel samples were quantified for the further method validation with satisfactory recoveries of 106.5 and 120.5%, respectively. This work proposed a sensitive, rapid, reliable and convenient analytical method for the potential study of trace and small molecular biological VOCs by the novel NAAA SPME fiber coating.     

Pomegranate By-Products as Natural Preservative to Prolong the Shelf Life of Breaded Cod Stick

This work evaluated the efficacy of pomegranate byproducts, specifically peel powder, as valid preservatives for food quality. Ready-to-cook cod sticks breaded with pomegranate peel powder were prepared. Shelf-life tests were conducted on breaded cod sticks during refrigerated storage (17 days) at 4 °C, monitoring the pH, microbiological and sensory quality. In addition, the nutritional quality of both the breaded and control samples was assessed. The results highlighted that active samples showed higher phenol and flavonoid content and higher antioxidant activity compared to the control fish, suggesting that pomegranate peel powder was responsible for a significant increase in cod stick nutritional quality. Furthermore, the cod stick active breading led to a delay in microbial growth without affecting the sensory properties; rather, it helped slow down the sensory attribute decline during the refrigerated storage. The data suggest that using pomegranate byproducts in breaded cod stick was effective in prolonging its shelf life, as well as improving its nutritional quality. Therefore, pomegranate peel powder can be considered as a potential resource as natural food preservative.     

Capillary-based fully integrated and automated system for nanoliter polymerase chain reaction analysis directly from cheek cells

A miniaturized, integrated and automated system based on capillary fluidics has been developed for nanoliter DNA analysis directly from cheek cells. All steps for DNA analysis, including injecting aqueous reagents and DNA samples, mixing the solutions together, thermal cell lysis, polymerase chain reaction (PCR), transfer and injection of PCR product, separation, sizing and detection of those products are performed in a capillary-based integrated system. A small amount of cheek cells collected by a plastic toothpick is directly dissolved in the PCR cocktail in a plastic vial or mixed on-line with a small volume of PCR cocktail (125 nl) in the capillary. After thermal cell lysis and PCR in a microthermal cycler, the DNA fragments are mixed with DNA size standards and transferred to a micro-cross for injection and separation by capillary gel electrophoresis. Programmable syringe pumps, switching valves, multiposition and freeze-thaw valves are used for microfluidic control in the entire system. This work establishes the feasibility of performing all the steps of DNA analysis from real samples in a capillary-based nanoliter integrated system.     

Determination of fluquinconazole, pyrimethanil, and clofentezine residues in fruits by liquid chromatography with ultraviolet detection

A simple method was developed for the determination of fluquinconazole, pyrimethanil, and clofentezine in whole fruit; peel; and pulp of mango, apple, and papaya. These compounds were extracted from fruit samples with a mixture of ethyl acetate-n-hexane (1 + 1, v/v). An aliquot (2 mL) of the extract was evaporated to near dryness under a stream of nitrogen, and the residue was dissolved with 2 mL methanol. The analysis was performed by means of liquid chromatography with ultraviolet detection at 254 nm using a gradient solvent system. The method was validated with fortified fruit samples at concentration levels of 0.05, 0.10, 0.20, and 0.50 mg/kg. Average recoveries (4-8 replicates) ranged from 80 to 95% with relative standard deviations between 3.5 and 12.7%. Detection limits ranged from 0.03 to 0.05 mg/kg for fruit pulp and 0.03 mg/kg for whole fruit. The quantitation limits ranged from 0.05 to 0.10 mg/kg for fruit pulp and 0.05 mg/kg for whole fruit. The analytical method was applied to fruit samples obtained from local markets.     

Residue determination of captan and folpet in vegetable samples by gas chromatography/negative chemical ionization-mass spectrometry

A gas chromatography/negative chemical ionization-mass spectrometry (GC/NCI-MS) method has been developed for the simultaneous determination of the fungicides captan and folpet in khaki (persimmon; flesh and peel) and cauliflower. Samples were extracted with acetone in the presence of 0.1 M zinc acetate solution in order to avoid degradation of fungicides and were purified using solid-phase extraction with divinylbenzene polymeric cartridges. Purified extracts were evaporated and dissolved in hexane prior to injection into the GC/NCI-MS system. Isotope-labeled captan and folpet were used as surrogate/internal standards, and quantification was performed using matrix-matched calibration. The method showed linear response in the concentration range tested (50-2500 ng/mL). The method was fully validated with untreated blank samples of khaki (flesh and peel) and cauliflower spiked at 0.05 and 0.5 mg/kg. Satisfactory recoveries between 82 and 106% and relative standard deviations lower than 11% in all cases (n = 5) were obtained. The limit of detection for both compounds were estimated to be 0.01 mg/kg. The developed method has been applied to treated and untreated samples collected from residue trials.     

闪蒸-气相色谱指纹图谱及系统聚类分析用于烟用香精香料的测定

采用闪蒸-气相色谱法(FE-GC)对不同黏度的烟用香精香料样品进行测定,考察了影响测定的主要因素。结果表明,0.40 mg样品在350 ℃下进行FE-GC分析,可以得到重现性良好的色谱图。与超声辅助液-液萃取-气相色谱法(ULLE-GC)分析结果的比较表明,FE-GC适用于不同黏度样品的分析。采用该法检测了中等黏度的烟用香精香料1184号8个批次样品,并建立其指纹图谱。利用系统聚类分析法可以明显区分1184号样品及掺兑10%～30%其他种类烟用香精香料的样品。FE-GC法简便、快速、灵敏,适用于不同黏度的烟用香精香料样品的检测和质量控制分析。     

穿山甲色谱指纹谱的建立及其在基源鉴别和质量等级区分方面的应用

中药穿山甲为鳞鲤科动物穿山甲的鳞甲,常用于治疗乳汁不通、痈肿疮毒、风湿痹痛等症,饮片来源上经常存在基源不清、等级不明的问题。针对以上问题,目前已发展出了多种分析方法,包括薄层色谱、质谱和DNA检测,但由于特异性低、设备成本高、步骤繁琐等不足,以上方法均不适合作为穿山甲质量监测的常规手段,而色谱指纹谱技术恰好可以弥补以上不足。该文旨在建立穿山甲的色谱指纹谱,并探讨其在等级及基源鉴别方面的可行性。穿山甲粉末经1 mol/L HCl加热水解得供试液,色谱柱采用Waters Symmetry 300 C18,以0.1%(v/v)三氟乙酸/水、0.1%(v/v)三氟乙酸/乙腈为流动相,280 nm为检测波长,采用梯度洗脱获得穿山甲指纹谱。经考察,该方法的精密度、日内日间重复性及样品稳定性均表现良好(RSD<5%)。以12批中华穿山甲一等品的平均指纹谱为对照指纹谱,同时确认17个共有峰,继而以共有峰绝对峰面积为原始数据,计算样品指纹谱与对照指纹谱的相似度。结果表明,中华穿山甲与其他动物来源穿山甲的相似度不高于0.776,体现了相似度评价在基源鉴别上的有效性;但不同等级穿山甲之间交叉严重,表明相似度评价在等级鉴别上的局限性。以24批不同等级中华穿山甲为分析对象建立判别分析模型,经十折交叉验证,模型无偏差正确率为95.83%,说明该模型在等级区分上可行性很高。该文通过建立穿山甲的色谱指纹谱,并结合不同数据处理方法验证了其在基源鉴别和等级区分方面的可行性,为保证穿山甲临床用药的科学性、准确性提供新的思路。     

发酵虫草菌粉及产品指纹图谱建立及多指标成分分析

《中国药典》收载的发酵虫草菌粉产品的质量标准中,规定以鸟苷、腺苷、尿苷的含量作为评价相关产品质量的标准.但除此之外,还有许多其他的核苷类成分对发酵虫草菌粉质量控制的影响尚未被探讨.为探究发酵虫草菌粉及产品质控指标选择的合理性,采用超高效液相色谱-紫外检测法对19批发酵虫草菌粉及产品中9种核苷成分(尿嘧啶、胞苷、鸟嘌呤、...     

LC-MS/MS determination of 2-(4-((2-(2S,5R)-2-Cyano-5-ethynyl-1-pyrrolidinyl)-2-oxoethylamino)-4-methyl-1-piperidinyl)-4-pyridinecarboxylic acid (ABT-279) in dog plasma with high-throughput protein precipitation sample preparation

As an effective DPP-IV inhibitor, 2-(4-((2-(2S,5R)-2-Cyano-5-ethynyl-1-pyrrolidinyl)-2-oxoethylamino)-4-methyl-1-piperidinyl)-4-pyridinecarboxylic acid (ABT-279), is an investigational drug candidate under development at Abbott Laboratories for potential treatment of type 2 diabetes. In order to support the development of ABT-279, multiple analytical methods for an accurate, precise and selective concentration determination of ABT-279 in different matrices were developed and validated in accordance with the US Food and Drug Administration Guidance on Bioanalytical Method Validation. The analytical method for ABT-279 in dog plasma was validated in parallel to other validations for ABT-279 determination in different matrices. In order to shorten the sample preparation time and increase method precision, an automated multi-channel liquid handler was used to perform high-throughput protein precipitation and all other liquid transfers. The separation was performed through a Waters YMC ODS-AQ column (2.0 x 150 mm, 5 microm, 120 A) with a mobile phase of 20 mm ammonium acetate in 20% acetonitrile at a flow rate of 0.3 mL/min. Data collection started at 2.2 min and continued for 2.0 min. The validated linear dynamic range in dog plasma was between 3.05 and 2033.64 ng/mL using a 50 microL sample volume. The achieved r(2) coefficient of determination from three consecutive runs was between 0.998625 and 0.999085. The mean bias was between -4.1 and 4.3% for all calibration standards including lower limit of quantitation. The mean bias was between -8.0 and 0.4% for the quality control samples. The precision, expressed as a coefficient of variation (CV), was < or =4.1% for all levels of quality control samples. The validation results demonstrated that the high-throughput method was accurate, precise and selective for the determination of ABT-279 in dog plasma. The validated method was also employed to support two toxicology studies. The passing rate was 100% for all 49 runs from one validation study and two toxicology studies.     

Determination of concentrations of U and Th in their mixed oxides by INAA methods

Uranium and thorium mixed oxides are being prepared using natural U and Th for studies on fuels for Advanced Heavy Water Reactors, wherein composition of U and Th is specific and requires strict control in terms their contents and homogeneity. Chemical quality control necessitates accurate and precise compositional characterization of the fuel material by a suitable analytical method. Among various analytical methods for U and Th, instrumental neutron activation analysis (INAA) is one of the best methods for their simultaneous determination without chemical dissolution and separation. INAA methods using reactor neutrons namely thermal NAA and epithermal NAA were standardized for the determination of U and Th in their mixed oxides. Standards, synthetic samples and U–Th mixed oxide samples, prepared in cellulose matrix, were irradiated at pneumatic carrier facility of Dhruva reactor as well as at self serve facility of CIRUS reactor under cadmium cover (0.5 mm). Radioactive assay was carried out using a 40% relative efficiency HPGe detector. Both activation and daughter products of ²³⁸U (²³⁹U and ²³⁹Np) and ²³²Th (²³³Th and ²³³Pa) were used for their concentration determination. The method was validated by analyzing synthetic samples of 6–48%U–Th mixed oxides. The standardized method was used for the concentration determination of U and Th in 4–30%U–Th mixed oxide samples. Results of U and Th concentrations including associated uncertainties obtained from the INAA methods are presented in this paper.     

A Validated, Specific Stability-Indicating RP-LC Method for Moxifloxacin and Its Related Substances

A validated, specific, stability-indicating reversed-phase liquid chromatographic method has been developed for quantitative analysis of moxifloxacin and its related substances in bulk samples and pharmaceutical dosage forms in the presence of degradation products and process-related impurities. Forced degradation studies were performed on bulk samples of moxifloxacin, in accordance with ICH guidelines, using acidic, basic, and oxidizing conditions, and thermal and photolytic stress, to show the stability-indicating power of the method. Significant degradation was caused by oxidative stress and by basic conditions; no degradation was observed under the other stress conditions. The method was optimized by analysis of the samples generated during the forced degradation studies and sample solutions spiked with the impurities. Good resolution between the analyte peak and peaks corresponding to process-related impurities and degradation products was achieved on a C₁₈ column with a simple linear mobile phase gradient prepared from aqueous sodium dihydrogen orthophosphate dihydrate containing triethylamine, pH adjusted to 3.0 with orthophosphoric acid, and methanol. Detection was performed at 240 nm. Limits of detection and quantification were established for moxifloxacin and its process related impurities. When the stressed test solutions were assayed against moxifloxacin working standard solution the mass balance was always between 99.3 and 100.1%, indicating the method was stability-indicating. The method was validated in accordance with ICH guidelines, and found to be suitable for checking the quality of bulk samples of moxifloxacin at the time of release of a batch and during storage (long term and accelerated stability testing was conducted).     

Characterization of aroma active compounds in fruit juice and peel oil of Jinchen sweet orange fruit (Citrus sinensis (L.) Osbeck) by GC-MS and GC-O

Gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O) were used to determine the aromatic composition and aroma active compounds of fruit juice and peel oil of Jinchen sweet orange fruit. Totals of 49 and 32 compounds were identified in fruit juice and peel oil, respectively. GC-O was performed to study the aromatic profile of Jinchen fruit juice and peel oil. A total of 41 components appeared to contribute to the aroma of fruit juice and peel oil. Twelve components were the odorants perceived in both samples. The aromatic compositions of fruit juice were more complex than that of peel oil. Ethyl butanoate, beta-myrcene, octanal, linalool, alpha-pinene, and decanal were found to be responsible for the aromatic notes in fruit juice and peel oil. Nineteen components have been perceived only in the juice and ten compounds were described as aromatic components of only the peel oil by the panelists. These differences lead to the different overall aroma between fruit juice and peel oil.     

Quality consistency evaluation of commercial transfer factor injections by chromatographic fingerprint combined with multivariate statistical analysis

The current quality control methods relying mainly on chromogenic reaction can hardly ensure the quality and safety of the biochemical drug with complex chemical composition. Therefore, a chromatographic fingerprint method was developed for the quality evaluation of a multicomponent biochemical drug, transfer factor injection. High‐performance liquid chromatography fingerprint was measured by using a C₁₈ column (250 × 4.6 mm, 5 µm) with a mobile phase composed of 0.1% trifluoroacetic acid–water and 0.085% trifluoroacetic acid–acetonitrile under gradient elution. The developed method was validated and was subsequently applied to 57 batches of commercial products which were sampled by National Drug Assessment Program. High‐resolution mass spectrometry analysis was performed on characteristic peaks of fingerprints, and a series of amino acids, nucleosides, and deoxynucleosides were identified. In the fingerprint assessments, principal component analysis and Hotelling T² analysis yielded the best results. The results generally indicated that there was a significant difference among products of batch‐to‐batch or from different manufacturers. Abnormal samples and its discriminatory components were also explored. In summary, the established fingerprinting method with multivariate statistical analysis could offer an efficient, reliable, and practical approach for quality consistency evaluation of transfer factor injection, providing a reference for the quality control of other multicomponent biochemical drugs.     

Quantitative analysis and chromatographic fingerprinting of the semen zizyphi spinosae by ultra-high-performance liquid chromatography coupled with diode-array detector

A simple and sensitive method was developed and validated for fingerprint analysis of semen zizyphi spinosae (SZS) and simultaneous determination of six flavonoids in SZS by ultra‐high‐performance liquid chromatography coupled to diode‐array detector (DAD). The analysis was performed on an Agilent ZORBAX Eclipse Plus C18 RRHD column. The column was maintained at 40°C and the eluents were monitored with DAD at 270 nm. A gradient elution of acetonitrile and water containing 20 mM sodium dihydrogen phosphate was used. The solvent flow rate was 0.4 mL/min. The method was validated. Standard calibration curves showed good linear behaviors (r=1.000) in the range of 0.33–201.00 μg/mL. Acceptable intra‐day precision (RSD<1.9%), inter‐day precision (RSD<4.0%), repeatability (RSD<4.1%) and recovery in the range of 97.4–104% were obtained. The validated method was successfully applied to obtain the chromatographic fingerprints and the contents of six flavonoids in 23 samples of SZS. The principal component analysis (PCA) had been applied for the chromatographic fingerprint analysis and quantitative analysis of six flavonoids to classify and discriminate the 23 samples of SZS. These results demonstrated that the method was very suitable in the analysis and quality control of SZS.     

Monitoring the quality consistency of Fufang Danshen Pills using micellar electrokinetic chromatography fingerprint coupled with prediction of antioxidant activity and chemometrics

A fast micellar electrokinetic chromatography fingerprint method combined with quantification was developed and validated to evaluate the quality of Fufang Danshen Pills, a traditional Chinese Medicine, which has been used in the treatment of cardiovascular system diseases, in which the tetrahedron optimization method was first used to optimize the background electrolyte solution. Subsequently, the index of the fingerprint information amount of I was performed as an excellent objective indictor to investigate the experimental conditions. In addition, a systematical quantified fingerprint method was constructed for evaluating the quality consistency of 20 batches of test samples obtained from the same drug manufacturer. The fingerprint analysis combined with quantitative determination of two components showed that the quality consistency of the test samples was quite good within the same commercial brand. Furthermore, the partial least squares model analysis was used to explore the fingerprint–efficacy relationship between active components and antioxidant activity in vitro, which can be applied for the assessment of anti‐oxidant activity of Fufang Danshen pills and provide valuable medicinal information for quality control. The result illustrated that the present study provided a reliable and reasonable method for monitoring the quality consistency of Fufang Danshen pills.     

Multicenter validation of PCR-based method for detection of Salmonella in chicken and pig samples

As part of a standardization project, an interlaboratory trial including 15 laboratories from 13 European countries was conducted to evaluate the performance of a noproprietary polymerase chain reaction (PCR)-based method for the detection of Salmonella on artificially contaminated chicken rinse and pig swab samples. The 3 levels were 1-10, 10-100, and 100-1000 colony-forming units (CFU)/100 mL. Sample preparations, including inoculation and pre-enrichment in buffered peptone water (BPW), were performed centrally in a German laboratory; the pre-PCR sample preparation (by a resin-based method) and PCR assay (gel electrophoresis detection) were performed by the receiving laboratories. Aliquots of BPW enrichment cultures were sent to the participants, who analyzed them using a thermal lysis procedure followed by a validated Salmonella-specific PCR assay. The results were reported as negative or positive. Outlier results caused, for example, by gross departures from the experimental protocol, were omitted from the analysis. For both the chicken rinse and the pig swab samples, the diagnostic sensitivity was 100%, with 100% accordance (repeatability) and concordance (reproducibility). The diagnostic specificity was 80.1% (with 85.7% accordance and 67.5% concordance) for chicken rinse, and 91.7% (with 100% accordance and 83.3% concordance) for pig swab. Thus, the interlaboratory variation due to personnel, reagents, thermal cyclers, etc., did not affect the performance of the method, which will be proposed as part of a developing international PCR standard.     

Validation of a carnation-specific gene, ANS, used as an endogenous reference gene in qualitative and real-time quantitative PCR for carnations

The validation of the anthocyanin synthase (ANS) gene as a carnation endogenous reference gene applicable both in classical and real-time PCR methods is a prerequisite for the development of PCR assays for genetically modified (GM) carnation detection. This is important due to the fact that GM carnation lines, developed by Florigene Pty Ltd, have been approved for commercialization. In this study, both methods were tested on 14 different carnation cultivars, and identical amplification products were obtained with all of them. No amplification products were observed with samples from 14 other plant species, which demonstrated that the system was specific to carnation. The results of Southern blot analysis confirmed that the ANS gene had a low copy number in the 10 tested carnation varieties. In qualitative and real-time PCR assays, the LOD values of 0.05 and 0.005 ng carnation DNA, respectively, were validated. Moreover, the real-time PCR system was validated with high PCR efficiency and linearity. Thus, the ANS gene had species specificity, low heterogeneity, and low copy number among the tested cultivars. These results provide evidence that the gene can be used as an endogenous reference gene of carnation, as well as in qualitative and quantitative PCR systems.