Chromatographic Fingerprint Analysis of Semen Ziziphi spinosae by HPLC-DAD Method

Abstract

Reliable, reproducible and valid fingerprint analysis methods using high-performance liquid chromatography-photodiode array detection (HPLC-DAD) for characteristic bioactive flavonoids and saponins of Semen Ziziphi spinosae (SZS) were developed and validated. HPLC separation of the chemical constituents of SZS was performed on an YMC-PACK ODS-A RP-18 column and detected at 270 and 204 nm for flavonoids and saponins, respectively. A mobile phase consisted of acetonitrile and 0.1% phosphoric acid aqueous solution was used with linear gradient elution. Using spinosin and jujuboside B as the reference markers of flavonoids and saponins respectively, 9 common fingerprint peaks of flavonoids and 10 common fingerprint peaks of saponins were specified based on the fingerprint analysis of 10 batches of SZS from different regions in China. The fingerprint analysis methods developed are reliable, reproducible and valid, and might be used as a more convenient approach for the species identification and quality monitoring and assessment of SZS.     

A UPLC‐MS/MS method for simultaneous determination of five flavonoids from Stellera chamaejasme L. in rat plasma and its application to a pharmacokinetic study

Stellera chamaejasme L. has been used as a traditional Chinese medicine for the treatment of scabies, tinea, stubborn skin ulcers, chronic tracheitis, cancer and tuberculosis. A sensitive and selective ultra‐high liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated for the simultaneous determination of five flavonoids (stelleranol, chamaechromone, neochamaejasmin A, chamaejasmine and isochamaejasmin) of S. chamaejasme L. in rat plasma. Chromatographic separation was accomplished on an Agilent Poroshell 120 EC‐C₁₈ column (2.1 × 100 mm, 2.7 μm) with gradient elution at a flow rate of 0.4 mL/min and the total analysis time was 7 min. The analytes were detected using multiple reaction monitoring in positive ionization mode. The samples were prepared by liquid–liquid extraction with ethyl acetate. The UPLC‐MS/MS method was validated for specificity, linearity, sensitivity, accuracy and precision, recovery, matrix effect and stability. The validated method exhibited good linearity (r ≥ 0.9956), and the lower limits of quantification ranged from 0.51 to 0.64 ng/mL for five flavonoids. The intra‐ and inter‐day precision were both <10.2%, and the accuracy ranged from −11.79 to 9.21%. This method was successfully applied to a pharmacokinetic study of five flavonoids in rats after oral administration of ethyl acetate extract of S. chamaejasme L.     

Rapid determination of fosetyl-aluminium in commercial pesticide formulations by high-performance liquid chromatography

An isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) method with diode array detection (DAD) was developed for the determination of aluminium tris(ethyl phosphonate) (fosetyl-aluminium, fosetyl-Al) in plant-protection products. The method involves extraction of the active ingredient by sonication of the sample with water and direct measurement by RPHPLC. The isocratic RP-HPLC method for the analysis of fosetyl-Al thus developed was then validated for specificity, linearity, precision, and accuracy. The chromatographic peak confirmation was performed by LC-MS using electron spray ionisation in the negative-ion mode. The repeatability of the method, expressed as relative standard deviation (RSD, %), was found to be 0.5 % and the limit of detection was 0.035 mg mL^?1. The average recoveries of the three fortification levels varied from 96.7 % to 100.6 % and the RSDs ranged between 2.6 % and 6.3 %. The precision of the method was also considered to be acceptable as the experimental repeatability relative standard deviation (RSD_r) was lower than the RSD_r, calculated using the Horwitz equation. The method is rapid, simple, accurate, cost-effective, and provides a new and reliable means for the analysis of fosetyl-Al in formulated products.     

Simultaneous determination of six active components by a single standard to determine multicomponents combined with fingerprint analysis for the quality control of Rhizoma Chuanxiong

To control the quality of Rhizoma Chuanxiong, a method based on high‐performance liquid chromatography method coupled with diode array detection was developed for the quantitative analysis of six active ingredients using a single standard to determine multi‐components and chemical fingerprint analysis for the first time. The separation was performed on an Agilent Zorbax SB‐C₁₈ column by gradient elution with methanol and aqueous phase (containing 0.5% glacial acetic acid) at a flow rate of 1.0 mL/min. The UV wavelength was set at 274 nm. This assay was fully validated with respect to precision, repeatability, and accuracy. All calibration curves showed good linearity (R²> 0.9994) within test ranges. The limit of detection and limit of quantification were lower than 0.01 and 0.03 μg/mL, respectively. The relative standard deviation for repeatability and the intermediate precision of six analytes were less than 1.6 and 2.5%, respectively, the overall recovery was 96.1–103.1%. In addition, fingerprint chromatography using hierarchical clustering analysis and similarity analysis was performed to differentiate and classify the samples. The method described here could provide a more comprehensive and reasonable scientific assessment of the quality of Rhizoma Chuanxiong. Therefore, the strategy is feasible, credible, and is easily and effectively adapted for evaluating the quality control of Rhizoma Chuanxiong.     

A comparative pharmacokinetic study of three flavonoids and three anthraquinones in normal and gastrointestinal motility disorders rat plasma after the oral administration of Wei‐Chang‐Shu tablet using high‐performance liquid chromatography–tandem mass spectrometry

A simple, fast and reliable high‐performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous quantification and pharmacokinetic study of three flavonoids (liquiritigenin, isoliquiritigenin and formononetin) and three anthraquinones (emodin, rhein and aloe‐emodin), which are the bioactive ingredients of Wei‐Chang‐Shu tablet found in rat plasma. After extraction by liquid–liquid extraction with ethyl acetate, chromatographic separation was achieved on an Agilent Zorbax SB‐C₁₈ column (4.6 × 150 mm, 5 μm) at a flow rate of 1 mL/min by gradient elution using 0.1% aqueous acetic acid and acetonitrile. The detection was performed using a triple quadrupole mass spectrometer equipped with electrospray ionization source in the negative ionization and selected reaction monitoring mode. Method validation was performed in terms of specificity, carryover, linearity (r > 0.99), intra−/inter‐day precision (1.0–10.1%), accuracy (relative error, <7.6%), stability (0.6–13.2%), extract recovery (74.9–91.9%) and matrix effect (89.1–109%). The lower limits of quantification of the six analytes varied from 0.92 to 10.4 ng/mL. The validated method was successfully applied to compare the pharmacokinetic properties of Wei‐Chang‐Shu tablet in normal rats and in rats with gastrointestinal motility disorders. The results indicated that there were obvious differences in the pharmacokinetic behavior between normal and model rats. This study will be helpful in the clinical application of Wei‐Chang‐Shu tablet.     

Validation of a new HPLC‐UV method for determination of the antibiotic linezolid in human plasma and in bronchoalveolar lavage

A rapid and selective HPLC‐UV method was developed for the quantification of linezolid (LNZ) in human plasma and bronchoalveolar lavage (BAL) at the concentrations associated with therapy. Plasma samples were extracted by solid‐phase extraction followed by evaporation to dryness and reconstitution in mobile phase solution. The chromatographic separation was carried out on a C₁₈ column with an isocratic mobile phase consisting of dihydrogen phosphate buffer 50 mm (pH 3.5) and acetonitrile (60:40 v/v). The detection was performed using a photodiode array. Under these conditions, a single chromatographic run could be completed within 12 min. The method was validated by estimating the precision and the accuracy for inter‐ and intra‐day analysis in the concentration range of 25–25600 ng/mL. The method was linear over the investigated range with all the correlation coefficients R > 0.999. The intra‐ and inter‐day precision was within 8.90% and the accuracy ranged from ?4.76 to +5.20%. This rapid and sensitive method was fully validated and could be applied to pharmacokinetic study for the determination of LNZ levels in human plasma and BAL samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid HPLC analysis of the antiepileptic lamotrigine and its metabolites in human plasma

A liquid chromatographic method with diode array detection (DAD) has been developed for the analysis of the antiepileptic agent lamotrigine (LTG) and its metabolites, lamotrigine 2-N-glucuronide and 2-N-methylated in plasma samples. The analytes were separated on a C8 RP column, using a mobile phase composed of methanol and a 0.45 mM, pH 3.5 phosphate buffer containing 0.17% triethylamine (24:76 v/v). Melatonin was used as the internal standard (IS). The DAD detector was set at 220 nm for the detection of all the analytes. A simple protein precipitation with methanol guaranteed high extraction yield values (>90%) and good purification from matrix interference. Good linearity was obtained in the 0.1-15.0 microg/mL range for LTG and lamotrigine 2-N-glucuronide and in the 0.1-2.0 microg/mL range for lamotrigine 2-N-methylated. The analytical method was validated in terms of precision, extraction yield, and accuracy. These assays gave RSD% values for precision always lower than 4.3% and mean accuracy higher than 80%. The method seems to be suitable for the analysis of plasma samples from patients treated with Lamictal.     

Sensitive and rapid high‐performance liquid chromatography tandem mass spectrometry method for estimation of fulvestrant in rabbit plasma

A rapid and sensitive high‐performance liquid chromatography and electrospray tandem mass spectrometry method was developed and validated for estimation of fulvestrant in rabbit plasma using liquid–liquid extraction. The separation and quantification of fulvestrant were achieved by reverse‐phase chromatography on a Sunfire C₁₈ column (50 × 2.1. i.d., 3.5 μm) with isocratic elution at a flow rate of 300 μL/min using norethistrone as an internal standard from 500 μL plasma sample. The method was validated over the concentration range from 0.092 to 16.937 ng/mL with a lower limit of detection of 0.023 ng/mL. The intra‐day and inter‐day accuracy and precision were within 10%. The recovery was 85 and 90% for fulvestrant and norethistrone respectively. The chromatographic run time was only 2.5 min. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of key flavonoid aglycones by means of nano‐LC for the analysis of dietary supplements and food matrices

A method for the analysis of flavonoids (myricetin, quercetin, naringenin, hesperitin, and kaempferol), with interesting bioactivity, has been developed and validated utilizing nano‐LC technique. In order to find optimal conditions, capillary columns (75 μm id × 10 cm) packed with different types of stationary phases, Kinetex® C18 core–shell (2.6 μm particle size), Hydride‐based RP‐C18 (sub‐2 μm particle size), and LiChrospher® 100 RP‐18 endcapped (5 μm particle size) were evaluated. The method was validated using Hydride‐based RP‐C18 stationary phase, with sub‐2 μm particle size. A good chromatographic performance, expressed in terms of repeatability (RSD, in the range 1.63–4.68% for peak area), column‐to‐column reproducibility (RSD not higher than 8.01% for peak area), good linearity and sensitivity was obtained. In particular limit of detection values between 0.07 and 0.31 μg/mL were achieved with on column focusing technique. The method was applied to the determination of studied flavonoids in dietary supplements as well as in food matrices. The amount of quercetin found in the first analyzed dietary supplement, was in agreement to the labeled content. In the other samples, where the content of flavonoids was not labeled, most of the studied flavonoids were determined in amounts somewhere comparable to those reported in literature.     

Determination of flavonoids in Tibetan herbal medicine of Pyrethrum tatsienense by HPLC with photodiode array detection

A simple, precise, and rapid method was developed for the determination of four flavonoids, apigenin (AP), tricin (TR), luteolin (LU), and 4′-methoxy-tricin (MTR) in the Tibetan herbal medicine of Pyrethrum tatsienense by the high-performance liquid chromatographic technique coupled with photodiode array detection (DAD). A C₁₈ reversed-phase column and a linear gradient elution system with acetonitrile-tetrahydrofuran-1% acetic acid as the mobile phase were employed. The method is validated in terms of the limit of detection (LOD), linearity, precision, and accuracy. The LODs were 48 ng/mL for AP, 75 ng/mL for TR, 88 ng/mL for LU, and 59 ng/mL for MTR, respectively. An excellent linear response was observed over the range specified for all analytes, as confirmed by the correlation coefficient with which from 0.9995 to 0.9999. The intraday RSDs ranged from 0.59 to 2.60%, and the interday RSDs were not higher than 0.05. The accuracy of the method was confirmed with an average recovery ranging from 95.9 to 100.0%. Since the extraction conditions can affect the extraction efficiency, nine different extracts of P. tatsienense were analyzed using different extraction solvents and extraction times. The contents of the four flavonoids in the extract of P. tatsienense were successfully determined within 18 min. The text was submitted by the authors in English.     

Quantification of flavonoid glycosides in an aqueous extract from the traditional Mongolian medicinal plant Dianthus versicolor FISCH

An HPLC‐diode array detection (DAD) method was established in order to investigate dried aerial parts of Dianthus versicolor FISCH. (Caryophyllaceae), a plant used in traditional Mongolian medicine against liver impairment. Aqueous extracts were separated on an Aquasil^® C₁₈ column with a linear gradient of acetonitrile (ACN) and water (adjusted to pH 2.8 with formic acid) as the mobile phase. LC‐IT‐MS facilitated the assignment of 26 flavonoids, among them a series of rare C‐glycosylated as well as O‐glycosylated derivatives, which are assumed to be the active principles. Quantification was performed and validated using isovitexin‐7‐O‐glucoside (saponarin) as the external standard. The method showed good linear behaviour (r² ≥0.9999) over the investigated concentration range (0.007–3.5 mg/mL). The good precision of the method allowed the successful qualitative and quantitative analysis of flavonoid‐glycosides in the aqueous extracts prepared from five different D. versicolor samples. Depending on the origin of the samples, the total flavonoid content was found to vary considerably from 0.41 to 3.30% in the aqueous extracts and from 0.07 to 0.57% in the crude drug. In addition, the relative composition of the various flavonoids was found to differ strongly. These results highlight the need for proper quality control for this herbal drug.     

Simultaneous determination of eight flavonoids in Sedum sarmentosum Bunge from different areas by UHPLC with triple quadrupole MS/MS

Sedum sarmentosum Bunge (SSB) is a traditional Chinese herbal medicine containing multiple components that has been extensively used clinically to treat chronic viral hepatitis and some inflammatory diseases. Total flavonoids are major pharmacologically active components of SSB. To gain a deeper understanding of SSB resources, we analyzed eight chemical constituents in 33 batches of SSB from 11 regions in China. An accurate, precise and sensitive ultra‐high‐performance liquid chromatography coupled with triple quadrupole electrospray tandem mass spectrometry method was developed for the determination of eight flavonoids in SSB. Under the optimized chromatographic conditions, good separation for the eight target components was obtained on an Agilent Zobax SB C₁₈ (50 × 2.1 mm, 5 μm) column within 4 min. The established methods were validated with good linearity (r ≥ 0.9988), precision (RSD ≤ 2.68%), stability (1.43–3.28%) and repeatability (1.14–2.89%). Moreover, the average recoveries were 95.91–100.68%, and the RSDs were 1.50–3.80%. In addition, the analytical conditions of UPLC–ESI–MS/MS provided better sensitivity with a shorter analysis time when compared with the HPLC–DAD method. Hierarchical clustering analysis and principal component analysis were performed to estimate and classify these samples based on the contents of the eight chemical constituents. This study provided the theoretical basis and scientific evidence for the development and utilization of SSB resources.     

A simple validated RP‐HPLC bioanalytical method for the quantitative determination of a novel valproic acid arylamide derivative in rat hepatic microsomes

A simple and specific bioanalytical method based on reversed‐phase high‐performance liquid chromatography (RP‐HPLC) coupled with ultraviolet detection was developed and validated for the determination of a novel valproic acid arylamide, N‐(2‐hydroxyphenyl)‐2‐propylpentanamide (HO‐AAVPA) in rat hepatic microsomes (a subcellular fraction containing phase I enzymes, especially cytochrome P450). The chromatographic separation was achieved using a reversed‐phase Zorbax SB‐C₁₈ column and a mobile phase of acetic acid in water (0.2% v/v) and acetonitrile (40:60 v/v) with a flow rate of 0.5 mL/min. The calibration curve was linear over the range of 882–7060 ng/mL (r² = 0.9987), and the lower limit of quantification and the lower limit of determination were found to be 882 and 127.99 ng/mL, respectively. The method was validated with excellent sensitivity, and intra‐day accuracy and precision varied from 93.79 to 93.12%, and from 2.12 to 4.36%, respectively. The inter‐day accuracy and precision ranged from 93.29 to 97.30% and from 0.68 to 3.60%, respectively. The recovery of HO‐AAVPA was measured between 91.36 and 97.98%. The assay was successfully applied to the analysis of kinetic metabolism and pharmacokinetic parameters in vitro by a substrate depletion approach. Copyright © 2014 John Wiley & Sons, Ltd.     

Highly sensitive UHPLC–DAD method for simultaneous determination of two synergistically acting antiepileptic drugs; levetiracetam and lacosamide: Application to pharmaceutical tablets and human urine

A simple and highly sensitive ultra‐high‐performance liquid chromatographic–diode array (UHPLC‐DAD) detection method was developed and validated for the simultaneous estimation of levetiracetam (LEV) and lacosamide (LAC). It was clinically proven that the combination of LEV and LAC exhibits a synergistic effect against refractory seizures in mice, which was the motivation for the analysis of this binary mixture both in bulk and in human urine samples. The binary mixture was resolved on a Hypersil BDS C₁₈ analytical column, utilizing a mobile phase of 0.050 mol L^?1 phosphate buffer (pH 5.60), methanol and acetonitrile in the ratio (80:10:10 v/v/v) using catechol as an internal standard. The mobile phase was pumped at a flow rate of 1.2 mL min^?1 with diode array detection at 205 nm for both drugs and 270 nm for IS. Calibration curves were linear with correlation coefficient >0.9990 over the studied concentration range of 0.1–70.0 μg mL^?1 for both drugs. The developed method was reproducible with low relative standard deviation values for intra‐ and inter‐day precision (<2.0%). Both drugs were determined in bulk, pharmaceutical formulations and human urine samples without any interference from complex matrices.     

Fingerprint analysis and multi‐ingredient quantitative analysis for quality evaluation of Xiaoyanlidan tablets by ultra high performance liquid chromatography with diode array detection

A rapid and sensitive ultra high performance liquid chromatography method with diode array detection was developed for the fingerprint analysis and simultaneous determination of seven active compounds in Xiaoyanlidan (XYLD) tablets. The chromatographic separations were obtained on an Agilent Eclipse plus C₁₈ column (50 × 2.1 mm id, 1.8 μm) using gradient elution with water/formic acid (1%) and acetonitrile at a flow rate of 0.4 mL/min. Within 63 min, 36 peaks could be selected as the common peaks for fingerprint analysis to evaluate the similarities among several samples of XYLD tablets collected from different manufacturers. In quantitative analysis, seven compounds showed good regression (R > 0.9990) within test ranges and the recovery of the method was within the range of 95.9–104.3%. The method was successfully applied to the simultaneous determination of seven compounds in six batches of XYLD tablets. These results demonstrate that the combination of chromatographic fingerprint analysis and simultaneous multi‐ingredient quantification using the ultra high performance liquid chromatography method with diode array detection offers a rapid, efficient, and reliable approach for quality evaluation of XYLD tablets.     

Quantitative determination of the group of flavonoids and saponins from the extracts of the seeds of Glinus lotoides and tablet formulation thereof by high-performance liquid chromatography

The total flavonoids and saponins of the seeds of Glinus lotoides in the crude extracts and tablet formulation thereof were quantified by reversed-phase high-performance liquid chromatographic (RP-HPLC) methods with UV detection. The saponins were analyzed after acid hydrolysis in 3 M HCI at 100 degrees C for 1 h. Vicenin-2 and mollugogenol B were isolated and used as reference substances for the quantification of total flavonoids and saponins, respectively. The identity and purity (> 97%) of the standards were confirmed by spectroscopic (UV, MS, and NMR) and chromatographic (HPLC) methods. The flavonoids and saponins of the crude extract of the seeds and tablet formulation were separated by RP-HPLC (Nucleosil RP-18 column, 250 mm x 4.6 mm) using linear gradient elution systems of acetonitrile-water-0.1 M H3PO4 for flavonoids and methanol-water for saponins. Satisfactory separation of the compounds was obtained in less than 30 and 25 min, for the flavonoids and saponins, respectively. The methods were validated for linearity, repeatability, limits of detection (LOD) and limits of quantification (LOQ). Repeatability (inter- and intra-day, n = 6 and 9, respectively) showed less than 2% relative standard deviation (RSD). The LOD and LOQ were found to be 0.075 and 0.225 mg/mL, respectively, for vicenin-2 and 0.027 and 0.082 mg/100 mL, respectively, for mollugogenol B. The content of flavonoids and saponins of six single tablets was between 95 and 103% for flavonoids and 94-98% for saponins. The validated HPLC methods were employed to standardize a fingerprint of a laboratory produced purified extract, which could be used as a secondary standard for the routine quality control. Accordingly, the purified extract was found to contain 21.3% flavonoids (vicenin-2, 10%) and 25.4% saponins (glinuside G, 14.2%).     

Determination of epacadostat,a novel IDO1 inhibitor in mouse plasma by LC–MS/MS and its application to a pharmacokinetic study in mice

A sensitive, specific and rapid LC‐ESI‐MS/MS method has been developed and validated for the quantification of epacadostat in mouse plasma using tolbutamide as an internal standard (IS) as per regulatory guidelines. Sample preparation was accomplished through a protein precipitation. Chromatographic separation was performed on an Atlantis dC₁₈ column using a binary gradient using mobile phase A (acetonitrile) and B (0.2% formic acid in water) at a flow rate of 0.90 mL/min. Elution of epacadostat and IS occurred at ~2.41 and 2.51 min, respectively. The total chromatographic run time was 3.2 min. A linear response function was established in the concentration range of 1.07–533 ng/mL. The intra‐ and inter‐day accuracy and precision were in the ranges of 1.81–12.9 and 3.80–11.1%, respectively. This novel method has been applied to a pharmacokinetic study in mice.     

LC–DAD and LC–ESI–MS Chromatographic Fingerprinting and Quantitative Analysis for Evaluation of the Quality of Huang-Lian-Jie-Du-Tang

LC–DAD coupled with electrospray tandem mass spectrometry (LC–ESI–MS–MS) has been used to evaluate the quality of the traditional Chinese medicine Huang-Lian-Jie-Du-Tang (HLJDT). Twenty-five chromatographic peaks were obtained from a C₁₈ analytical column by gradient elution with acetonitrile and formate buffer (containing 0.5% formic acid) at a flow rate of 1.0 mL min^?1. The linearity, precision, and accuracy of the data obtained were acceptable. Thirteen components were identified by ESI–MS, and seven of these were quantitatively analyzed by LC–DAD. The method was used to analyze ten batches of HLJDT, and both chromatographic fingerprints and quantitative data were used to evaluate the quality of the HLJDT. It was concluded that this LC–DAD–ESI–MS method enables more fully validated and complete evaluation and monitoring of the quality of HLJDT.