Waters Acquity Ultra-performance Liquid Chromatography Research Articles

Analysis of Secondary Metabolites of Elaeagnus Angustifolia Leaves Based on UPLC-Q-TOF-MS.

The leaves of Elaeagnus angustifolia, belonging to the Elaeagnaceae Juss. family, are known for their medicinal properties for relieving cough and asthma, as well as treating dysentery and diarrhea. To establish a rapid qualitative method for the detection of secondary metabolites in leaves of Elaeagnus angustifolia, including the identification and analysis of various secondary metabolites in leaves of Elaeagnus angustifolia. Samples were separated using a Waters ACQUITY H-Class ultra-performance liquid chromatography (UPLC) system (FTN autosampler, quaternary LC pump) and ACQUITY UPLC® BEH C18 column (1.7 μm, 2.1 mm× 100 mm). The flow rate was set to 0.4 mL/min, the injection volume was 1.0 μL, and the column temperature was set to 45 °C. The mobile phase was methanol (A) with) -0.1% formic acid in water (B). Samples were analyzed by quadrupole time-of-flight mass spectrometry (Q-TOF-MS). A total of 182 different secondary metabolites were detected from 10 varieties of leaves of Elaeagnus angustifolia, including 77 flavonoids, 20 steroids, 7 alkaloids, 15 amino acids, 18 organic acids, and 45 other compound types. A method for the rapid analysis of leaves of Elaeagnus angustifolia by UPLC-Q-TOF-MS was established, and the secondary metabolites in leaves of Elaeagnus angustifolia were identified. The enrichment of secondary metabolites in leaves of different varieties of Elaeagnus angustifolia was clarified. The UPLC-Q-TOF-MS method is very fast and possesses a high degree of selectivity, precision, and sensitivity. These findings provide a reliable foundation for the development of medicinal resources derived from Elaeagnus angustifolia leaves.

Development of a Stability Indicating UPLC Method for the Determination of Tirbanibulin in Bulk and Its Pharmaceutical Dosage Form.

The primary goal of this study was to create and validate a simple, precise, sensitive, and accurate ultra-performance liquid chromatography (UPLC) method for estimating tirbanibulin in pure and dosage form. A UPLC technique was developed using a Waters Acquity UPLC Phenyl (100 x 2.1 mm, 1.7 µm) column. The developed technique was validated in accordance with the International Conference on Harmonization (ICH) guidelines. Tirbanibulin was separated chromatographically with high resolution using the mobile phase acetonitrile: buffer (30:70 v/v) at 0.5 mL/min, 5 µL injection volume, and 220 nm wavelength. The validated technique was found to be linear in the 1-15 µg/mL range. The detection and quantification limits for tirbanibulin were 0.03 and 0.1 µg/mL, respectively. The percentage relative standard deviation was less than 2%, demonstrating the precision of the developed technique. Furthermore, the recovery rate was nearly 100%, confirming the accuracy of the method. Minor modifications to the chromatographic conditions demonstrated the robustness of the method. The developed analytical method was precise, simple, reproducible, and sensitive. Consequently, it can be used to determine tirbanibulin.

Levels of organophosphate flame retardants and their metabolites among 391 volunteers in Taiwan: difference between adults and children.

Organophosphate flame retardants (OPFRs) are ubiquitous in the environment. The compositions and concentrations of different OPFRs metabolites vary in different environments depending on different human activities. The objective of the present study was to evaluate the exposure of different age groups to OPFRs in Taiwan. Volunteers provided urine samples and responded to questionnaires including demographic factors, underlying disease, lifestyle information, and occupation from October 2021 to January 2022. OPFR measurements were performed using a Waters Acquity Ultra-Performance Liquid Chromatography system coupled with a Waters Xevo TQ-XS mass spectrometer. A total of 391 volunteers (74 children and 317 adults) were enrolled in this study. The concentrations (presented as μg/g creatinine) of bis(1,3-dichloro-2-propyl) phosphate (BDCPP, p = 0.029) and tri-n-butyl phosphate (TNBP, p = 0.008) were higher in the adult group, while the concentrations of bis-2-chloroethyl phosphate (BCEP, p = 0.024), diphenyl phosphate (DPHP, p < 0.001), tris(1,3-dichloro-2-propyl) phosphate (TDCPP, p = 0.009), and Tris(2-butoxyethyl) phosphate (TBEP, p = 0.007) were higher in the child group. Compared with school age children (>6 years), the concentration of di(2-n-butoxyethyl) phthalate (DBEP, 1.14 vs. 0.20 μg/g creatinine, p = 0.001), DPHP (1.23 vs. 0.54 μg/g creatinine, p = 0.036), TBEP (1.63 vs. 0.29 μg/g creatinine, p < 0.001), and the sum of OPFR metabolites (ΣOPFRs, 6.58 vs. 2.04 μg/g creatinine, p < 0.001) were statistically higher in preschool-aged children. After adjusting for confounding factors, pre-school age [odds ratio (OR): 4.579, 95% confidence interval (CI): 1.389-13.115] and current smoker (OR: 5.328, 95%CI: 1.858-14.955) were independently associated with the risk of ΣOPFRs higher than 90 percentile. This study revealed the distribution of different OPFRs metabolites in children and adults. DBEP, DPHP, TBEP, and ΣOPFR were higher in preschool-aged children. Pre-school age and current smoking status were independent risk factors for ΣOPFRs higher than 90 percentile.

Simultaneous determination of five indole/indazole amide-based synthetic cannabinoids in electronic cigarette oil by ultra performance liquid chromatography

Synthetic cannabinoids (SCs), which are considered some of the most widely abused new psychoactive substances available today, are much more potent than natural cannabis and display greater efficacy. New SCs can be developed by adding substituents such as halogen, alkyl, or alkoxy groups to one of the aromatic ring systems, or by changing the length of the alkyl chain. Following the emergence of the so-called first-generation SCs, further developments have led to eighth-generation indole/indazole amide-based SCs. Given that all SCs were listed as controlled substances on July 1, 2021, the technologies used to detect these substances must be quickly improved. Due to the sheer number of SCs, the chemical diversity and the fast update speed, it is challenging to determine and identify the new SCs. In recent years, several types of indole/indazole amide-based SCs have been seized, but systematic research on these compounds remains limited. Therefore, developing rapid, sensitive, and accurate quantitative methods to determine new SCs are of great importance. Compared with high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC) shows higher resolution, better separation efficiency, and faster analysis speeds; thus, it can meet the demand for the quantitative analysis of indole/indazole amide-based SCs in seized materials. In this study, a UPLC method was developed for the simultaneous determination of five indole/indazole amide-based SCs, including N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-butyl-1H-indazole-3-carboxamide (ADB-BUTINACA), methyl 2-(1-(4-fluorobutyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoate (4F-MDMB-BUTICA), N-(1-methoxy-3,3-dimethyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (5F-MDMB-PICA), methyl 3,3-dimethyl-2-(1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido)butanoate (MDMB-4en-PINACA), and N-(adamantan-1-yl)-1-(4-fluorobutyl)-1H-indazole-3-carboxamide (4F-ABUTINACA) in electronic cigarette oil; these SCs have been detected with increasing frequency in seized materials in recent years. The main factors influencing the separation and detection performance of the proposed method, including the mobile phase, elution gradient, column temperature, and detection wavelength, were optimized. The proposed method successfully quantified the five SCs in electronic cigarette oil via the external standard method. The samples were extracted using methanol, and the target analytes were separated on a Waters ACQUITY UPLC CSH C18 column (100 mm×2.1 mm, 1.7 μm) at column temperature of 35 ℃ and flow rate of 0.3 mL/min. The injection volume was 1 μL. The mobile phase consisted of acetonitrile and ultrapure water, and gradient elution was employed. The detection wavelengths were 290 and 302 nm. The five SCs were completely separated within 10 min under optimized conditions and showed good linear relationships between 1-100 mg/L, with correlation coefficients (r2) of up to 0.9999. The limits of detection (LOD) and quantification (LOQ) were 0.2 and 0.6 mg/L, respectively. Precision was determined using standard solutions of the five SCs at mass concentrations of 1, 10, and 100 mg/L. The intra-day precision (n=6) was <1.5%, and the inter-day precision (n=6) was <2.2%. Accuracy was determined by spiking electronic cigarette oil with low (2 mg/L), moderate (10 mg/L), and high (50 mg/L) levels of the five SCs, with six replicates per determination. The recoveries of the five SCs were 95.5%-101.9%, and their relative standard deviations (RSDs, n=6) were 0.2%-1.5%, with accuracies ranging from -4.5% to 1.9%. The proposed method showed good performance when applied to the analysis of real samples. It is accurate, rapid, sensitive, and effective for the determination of five indole/indazole amide-based SCs in electronic cigarette oil. Thus, it satisfies the requirements for practical determination and provides a reference for the determination of SCs with similar structures by UPLC.

Cellular toxicity and pharmacokinetic study of butachlor by ultra-performance liquid chromatography-tandem mass spectrometry based on tandem mass spectrometry cubed technique.

Butachlor is an aromatic amide compound that plays a role as a herbicide, a xenobiotic, and an environmental contaminant. The aim of this work was to develop a highly selective and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method based on the tandem mass spectrometry cubed technique to determine butachlor in a biological matrix. Butachlor and internal standard acetochlor were separated on a Waters Acquity ultra-performance liquid chromatography BEH C18 column (2.1 × 50mm, 1.7μm) with gradient elution using 0.1% formic acid aqueous solution (A) and acetonitrile (B) as mobile phases. The transitions selected for tandem mass spectrometry cubed quantitative analysis in positive ion mode were: for butachlor, mass-to-charge ratio 312.2→238.1→162.1; for acetochlor, mass-to-charge ratio 270.1→224.0→148.1. The total running time for each sample was 5.5 min. The ultra-performance liquid chromatography-tandem mass spectrometry cubed method showed a linear relationship (R2 ≥ 0.995) in the concentration range of 0.5-100ng/ml. The intra and interday accuracies are within the range of -10.6%-4.3% and precisions are between 4.48% and 13.14%. The novelty of the method is the use of tandem mass spectrometry cubed scanning mode, which improves selectivity and sensitivity. The results indicated that butachlor was cellular toxic. The safety of butachlor should be considered when it is used as a herbicide.

Steroidogenesis in Peripheral and Transition Zones of Human Prostate Cancer Tissue.

The peripheral zone (PZ) and transition zone (TZ) represent about 70% of the human prostate gland with each zone having differential ability to develop prostate cancer. Androgens and their receptor are the primary driving cause of prostate cancer growth and eventually castration-resistant prostate cancer (CRPC). De novo steroidogenesis has been identified as a key mechanism that develops during CRPC. Currently, there is very limited information available on human prostate tissue steroidogenesis. The purpose of the present study was to investigate steroid metabolism in human prostate cancer tissues with comparison between PZ and TZ. Human prostate cancer tumors were procured from the patients who underwent radical prostatectomy without any neoadjuvant therapy. Human prostate homogenates were used to quantify steroid levels intrinsically present in the tissues as well as formed after incubation with 2 µg/mL of 17-hydroxypregnenolone (17-OH-pregnenolone) or progesterone. A Waters Acquity ultraperformance liquid chromatography coupled to a Quattro Premier XE tandem quadrupole mass spectrometer using a C18 column was used to measure thirteen steroids from the classical and backdoor steroidogenesis pathways. The intrinsic prostate tissue steroid levels were similar between PZ and TZ with dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT), pregnenolone and 17-OH-pregnenolone levels higher than the other steroids measured. Interestingly, 5-pregnan-3,20-dione, 5-pregnan-3-ol-20-one, and 5-pregnan-17-ol-3,20-dione formation was significantly higher in both the zones of prostate tissues, whereas, androstenedione, testosterone, DHT, and progesterone levels were significantly lower after 60 min incubation compared to the 0 min control incubations. The incubations with progesterone had a similar outcome with 5-pregnan-3,20-dione and 5-pregnan-3-ol-20-one levels were elevated and the levels of DHT were lower in both PZ and TZ tissues. The net changes in steroid formation after the incubation were more observable with 17-OH-pregnenolone than with progesterone. In our knowledge, this is the first report of comprehensive analyses of intrinsic prostate tissue steroids and precursor-driven steroid metabolism using a sensitive liquid chromatography-mass spectrometry assay. In summary, the PZ and TZ of human prostate exhibited similar steroidogenic ability with distinction in the manner each zone utilizes the steroid precursors to divert the activity towards backdoor pathway through a complex matrix of steroidogenic mechanisms.

STABILITY INDICATING UPLC METHOD FOR ESTIMATION OF METFORMIN HYDROCHLORIDE AND NATEGLINIDE SIMULTANEOUSLY IN THE PRESENCE OF STRESS DEGRADATION PRODUCTS

A novel Ultra Performance Liquid Chromatography (UPLC) method was developed and validated for the simultaneous determination of antidiabetic drugs metformin hydrochloride and nateglinide. The method was developed using a Waters ACQUITY UPLC SB C18 (100 × 2.1 mm, 1.8 μm) column. The mobile phase consisting of 0.01 % potassium dihydrogen phosphate buffer (pH 5.8): acetonitrile (50: 50 V/V) was used throughout the analysis. The flow rate was 0.3 mL/min, the injection volume was 1.0 μL, column temperature was 30 0C, run time 3 min and detection was carried at 238 nm using a TUV detector. The retention times of metformin hydrochloride and nateglinide were found to be 1.28 1.71 min, respectively. Metformin hydrochloride and nateglinide were found to be linear over the concentration range of 125-750 and 15-90 μg/mL. The limit of detection and the limit of quantification for metformin hydrochloride were found to be 0.22 and 0.68 μg/mL, respectively, and, for nateglinide, 0.02 and 0.6 μg/mL, respectively. Developed method was validated as per ICH guidelines. The specificity of the method was confirmed by forced degradation study. The suggested method is suitable for determination of metformin hydrochloride and nateglinide in bulk and pharmaceutical dosage forms.

Simultaneous Determination of the Flavonoids and Limonoids in Citrus junos Seed Shells Using a UPLC-DAD-ESI/MS

Citrus junos seeds (CS) have been traditionally used for the treatment of cancer and neuralgia. They are also used to manufacture edible oil and cosmetic perfume. A large amount of CS shells without oil (CSS) are discarded after the oil in CS is used as foods or herbal remedy. To efficiently utilize CSS as a by-products, it needs to be studied through chemical analysis. Therefore, we developed an ultra-performance liquid chromatography (UPLC)-diode array detection (DAD) method for simultaneous determination and quantitative analysis of five components (two flavonoids and threes limonoids) in CSS. A Waters Acquity UPLC HSS T3 column C18 (2.1 × 100 mm, 1.8 μm) was used for this separation. It was maintained at 40℃. The mobile phase used for the analysis was distilled water and acetonitrile with gradient elution. To identify the quantity of the five components, a mass spectrometer (MS) with an electrospray ionization (ESI) source was used. The regression equation showed great linearity, with correlation coefficient ≥ 0.9912. Limits of detection (LOD) and limits of quantification (LOQ) of the five compounds were 0.09 - 0.13 and 0.26 - 0.38 μg/mL, respectively. Recoveries of extraction ranged from 97.45% to 101.91%. Relative standard deviation (RSD) values of intra- and inter-day precision were 0.06 - 1.15% and 0.19 - 0.25%, respectively. This UPLC-DAD method can be validated to simultaneously analyze quantities of marker flavonoids and limonoids in CSS.

Simultaneous Determination of Methocarbamol and Paracetamol in the Presence of Three Related Substances by Ultra Performance Liquid Chromatography

Introduction:A rapid, and accurate Ultra Performance Liquid Chromatography (UPLC) method was developed to simultaneously analyze Methocarbamol, Paracetamol and the related substancesMaterials and Methods:Waters ACQUITY UPLC® BEH Phenyl C18 column was used in conjunction with UV detection at 225nm. Gradient elution with 0.05M, pH 6 phosphate buffer and acetonitrile flow at 0.3mL /min rate were used to separate the substances. The retention times for 4-Aminopheno, Paracetamol, Guaifenesin, Methocarbamol, and 4-Chloroacetanilide were 1.319 minute, 2.224 minute, 4.467 minute, 4.769 minute and 5.433 minute respectively. The concentration was linear in the range of 2-100 µg/ml for Methocarbamol, and 1-100 µg/mL for Paracetamol. The percentage recoveries were between 99.28±1.23% to 100.57±0.99% for Methocarbamol, and between 99.08±1.23% to 101.23±1.39% for Paracetamol.Results and Discussion:The validated optimal protocol is robust and accurate for simultaneous analysis of Methocarbamol, Paracetamol and the related substances, applicable for bulk powder as well as pharmaceutical formulation.Conclusion:In this paper, a highly sensitive, accurate, and precise UPLC method with UV-Vis detection was developed and validated for quality control of MET and PAR in bulk as well as in pharmaceutical preparations.

Quality research of Puerariae Lobatae Radix from different habitats with UPLC fingerprint and determination of multi-component content

To establish ultra performance liquid chromatography( UPLC) fingerprint of Puerariae Lobatae Radix from different habitats and simultaneously determine the contents of six isoflavonoids. The UPLC fingerprint analysis and content determination were performed on a Waters ACQUITY UPLC BEH C_(18)( 2. 1 mm×50 mm,1. 7 μm) chromatographic column,with acetonitrile-0. 05% formic acid as mobile phase for gradient elution. The detection wavelength was set at 250 nm; the flow rate was 0. 2 mL·min~(-1); the column temperature was 30 ℃ and the injection volume was 2 μL. Similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine( TCM) was adopted; principal component analysis( PCA) and discriminant analysis by partial least square method( PLS-DA) in Simca-P software were used to identify the differential components in samples from three habitats. The similarity was over 0. 90 in 29 batches of samples,indicating good consistency of the samples. The samples were clustered into 3 categories by PCA and PLS-DA,and six differential components such as puerarin apioside,daidzin,and isoflavoues aglycone were found. The determination results of 6 isoflavones,including 3'-hydroxy puerarin,puerarin,3'-methoxy puerarin,puerarin apioside,daidzin,and isoflavoues aglycone,showed that the content of the same component and the fluctuation range between different components were all different among different habitats. The total content of 6 isoflavones from different regions was Anhui 11. 21% >Henan 10. 97% >Shannxi 9. 38%. The establishment of UPLC fingerprint combined with simultaneous determination of 6 active components provides a more comprehensive reference for quality control and quality evaluation of Puerariae Lobatae Radix.

Comparative Pharmacokinetic Study of Mangiferin in Normal and Alloxan-Induced Diabetic Rats after Oral and Intravenous Administration by UPLC-MS/MS

Backgrounds: Diabetes mellitus (DM)-induced morphological and/or functional complications may alter the pharmacokinetic profiles of mangiferin. This study aims to compare pharmacokinetic profiles of mangiferin in normal and alloxan-induced diabetic rats after oral and intravenous administration. Methods: Mangiferin was administered orally (10 mg/kg) and intravenously (2 mg/kg) to normal and alloxan-induced diabetic Sprague-Dawley (SD) rats (n = 8). Blood samples were collected at different time points post-dose. Mangiferin and esculentoside (internal standard) were analyzed by Waters Acquity ultra-performance liquid chromatography system and TSQ Quantum Ultra triple quadrupole mass spectrometer (UPLC-MS/MS). Results: Mangiferin in normal and alloxan-induced diabetic rats experienced serious first-pass effect, which resulted in 1.71 and 0.80% of oral bioavailability respectively. Meanwhile, mangiferin was predominantly restricted to blood but not extensively distributed to organ tissues after intravenous administration. Compared with normal rats, the diabetic condition induced 53.26 and 50.90% decreases in C_max and AUC_0–t, respectively, for mangiferin after oral administration, and 63.08% decreases in C_max after intravenous administration. Conclusions: Compared to normal rats, pharmacokinetic parameters of mangiferin were altered in diabetic condition induced by alloxan. The findings might help to provide useful evidence for modeling of diabetic rats and the clinical applications of mangiferin.

A Novel Ultra-High Performance Liquid Chromatography Method for the Determination of Erdosteine, Related Impurities and Degradation Products in New Effervescent Tablets

A novel and automated, stability-indicating, reversed phase ultra performance liquid chromatography (UPLC) method was developed and validated for the quantitative determination of erdosteine, its known impurities and two novel degradation products in a new pharmaceutical dosage form (effervescent tablets). The chromatographic separations were performed on a Waters Acquity UPLC HSS T3, 1.8 µm (2.1 mm × 150 mm, I.D.) stainless steel column. The mobile phase consisted of 0.1% TFA in water and methanol under gradient elution conditions, at a flow rate of 0.29 mL/min, for the assay and impurities analysis. UV detection was set at a wavelength of 238 nm. Erdosteine raw material, placebo and effervescent tablets were subjected to forced degradation. The new degradation products (labeled OX1 and OX2) were found after oxidative treatment and characterized by ultra performance liquid chromatography mass spectrometry. The validation parameters such as linearity, limit of detection (LOD) and quantification (LOQ), accuracy, precision, specificity and robustness were highly satisfactory for all analyzed compounds. LOD (0.020 and 0.011–0.385 µg/mL for erdosteine and impurities, respectively) and LOQ values show the high sensibility of the method. Specificity of the method was confirmed by testing the matrix components. The validated method demonstrated to be suitable for routine quality control purposes and for routine stability studies of erdosteine in effervescent formulations.

Quality control of Dandeng Tongnao Ruanjiaonang based on UPLC fingerprint combined with chemical pattern recognition

To establish the ultra performance liquid chromatography (UPLC) fingerprint of Dandeng Tongnao Ruanjiaonang and conduct a systemic, comprehensive quality evaluation of the drug by combining with a chemical pattern recognition method. In this study, Waters UPLC ultra-high performance liquid chromatography instrument and ACQUITY UPLCHSS T3 chromatographic colum n were employed to perform the separation with acetonitrile-0.1% formic acid aqueous solution as the mobile phase for gradient elution; and the detection wavelength was set at 256 nm to establish the UPLC fingerprint of 10 batches of Dandeng Tongnao Ruanjiaonang. Then, the further quality assessment of the drug was carried out by similarity evaluation, Cluster Analysis(CA), Principal Component Analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Finally, 77 peaks were recognised as common peaks in the fingerprint, and 15 peaks of them were identified using standard references. The similarity value of these 10 batches of drugs was all above 0.960, indicating a relatively stable quality. But minor differences were still discovered between the batches of the drug by CA and PCA. Finally, 6 common peaks were recognised as the quality makers using OPLS-DA method. The analysis method established in this study was scientific, accurate, reliable and simple; fingerprint combined with chemical pattern recognition technique can be used to systematically and comprehensively evaluate the drug quality of Dandeng Tongnao Ruanjiaonang; what's more, it could also provide a reference for the quality control of traditional Chinese medicine and its preparations at the same time.

UPLC fingerprint and multi-components determination of three processed products of Rhizome of Curcuma wenyujin

To analyse the quality of three processed products of Rhizome of Curcuma wenyujin by establishing an ultra performance liquid chromatography (UPLC) method for simultaneously determining five sesquiterpene components in three processed products of rhizome of C. wenyujin and establishing UPLC fingerprints. Component determination was achieved on Waters ACQUITY UPLC BEH C₁₈ column（2.1 mm×50 mm, 1.7 μm), with acetonitrile-water as mobile phase for gradient elution. The flow rate was 0.3 mL·min⁻¹; column temperature was 30 °C; the detection wavelength was set at 214 nm and injection volume was 1 μL. The similarity was analyzed with "Similarity Evaluation System for Chromatographic Fingerprint of Chinese Materia Medica (2012.130723)", and hydrophobic cluster analysis (HCA), principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were conducted by using simca-p14.1 software to investigate the differences in components among these three kinds of processed products. The curzerene, curdione, curcumol, germacrone, furanodiene and β-elemene showed good linearity relationship with chromatographic peak area within the ranges of 10.8-320（r=0.999 9）, 10.36-259（r=0.998 1）, 10.54-263.5（r=0.999 3）, 30.2-755（r=0.999 6）and 34.38-862（r=0.999 9）mg·L⁻¹, respectively; their average recoveries were 98.75%, 98.69%, 98.63%, 99.76% and 99.57% respectively, with RSD of 2.67%, 1.47%, 1.29%, 2.54% and 0.87% respectively. The similarity of 30 batches of samples was larger than 0.9, indicating good consistency of the samples. The samples can be clearly classified into three categories for HCA, PCA and OPLS-DA pattern recognition, the differential chromatographic peak among three processed products was found respectively. The results showed that the pharmacology basis had changed obviously after processing of Rhizome of C. wenyujin, so it can provide the scientific basis for rational clinical application and establishing quality standards of three processed products of Rhizome of curcuma wenyujin.

Automated Integration of a UPLC Glycomic Profile.

Ultra-performance liquid chromatography (UPLC) is the established technology for accurate analysis of IgG Fc N-glycosylation due to its superior sensitivity, resolution, speed, and its capability to provide branch-specific information of glycan species. Correct and cost-efficient preprocessing of chromatographic data is the major prerequisite for subsequent analyses ranging from inference of structural isomers to biomarker discovery and prediction of humoral immune response from characterized changes in glycosylation. The complexity of glycomic chromatograms poses a number of challenges for developing automated data annotation and quantitation algorithms, which frequently necessitated manual or semi-manual approaches to preprocessing, most notably to peak detection and integration. Such procedures are meticulous and time-consuming, and may be a source of confounding due to their dependence on human labelers. Although liquid chromatography is a mature field and a number of methods have been developed for automatic peak detection outside the area of glycomics analysis, we found that hardly any of them are suitable for automatic integration of UPLC glycomic profiles without substantial modifications. In this chapter, we illustrate practical challenges of automatic peak detection of UPLC glycomics chromatograms. We outline a robust, semi-supervised method ACE (Automatic Chromatogram Extraction) for automated alignment and detection of glycan peaks in chromatograms, developed by Pharmatics Limited (UK) in collaboration with Genos Limited (Croatia). Application of the tool requires minimal human interference, which results in a significant reduction in the time and cost of IgG glycomics signal integration using Waters Acquity UPLC instrument (Milford, MA, USA) in several human cohorts with blind technical replicas.

UPLC Method for Determination of Eight Flupirtine Impurities in Bulk Substance and Their Characterization by 1H NMR and ESI–MS

A novel and simple ultra performance liquid chromatography (UPLC) method was developed for the simultaneous quantification of flupirtine and its related impurities in active pharmaceutical ingredient. The analysis was carried out on Waters Acquity UPLC high strength silica C18 (100 × 2.1 mm) 1.8 µm column with 0.19 mL min−1 flow rate in gradient mode at 347 nm wavelength. The elution of all nine components was established within 30 min. The retention time of the flupirtine peak was observed at about 7 min. The proposed method was validated as per the current international conference on harmonization guidelines for specificity, precision, linearity, accuracy, range, limit of detection, limit of quantification, robustness and solution stability parameters. The exhaustive method study was performed to indicate the stability-indicating nature of the method. Furthermore, the work has been extended to the characterization of flupirtine and its related impurities by using proton nuclear magnetic resonance spectroscopy and electron source ionization mass spectroscopy.

Characteristic Fingerprint Analysis of Mallotus philippinensis by Ultra Performance Liquid Chromatography Electrospray Ionization Mass Spectrometry

The developing countries mostly trust on traditional remedies, which include the use of different plant extracts or the bioactive phytoconstituents. For this purpose, analysis such as chemical fingerprinting strongly represents one of the best possibilities in searching of new economic and therapeutically effective plants for medicine. Mallotus philippinensis Muell. Arg (Euphorbiaceae) is a large genus of the trees and shrubs mainly distributed in the tropical and subtropical regions and are reported to have widespread range of pharmacological activities. A new, simple and rapid ultraperformance liquid chromatography (UPLC) method with photodiode array (PDA) detector has been developed. Further confirmation was performed by electrospray ionization mass spectrometry (ESI-MS) for the chemical fingerprint analysis in extracts of Mallotus philippinensis. The chromatographic separations were obtained on a Waters ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm) column using a gradient elution with 0.1% (v/v) formic acid/water and acetonitrile as mobile phase at a flow rate of 0.4 mL/min. The UPLC-PDA-ESI-MS characteristic fingerprints were established and 7 characteristic peaks were identified along with 5 unknown peaks within 4.5 min by comparing the retention times, λ max (nm), and MS spectra with the literature data. Therefore, this fingerprint analysis method can be applied for the identification and quality control of Mallotus philippinensis.

Development and validation of an analytical method for quantification of 15 non-tricyclic antidepressants in serum with UPLC-MS/MS

Non-tricyclic antidepressants are often encountered in overdose cases, and for some of them therapeutic monitoring might be warranted. Therefore, we developed and validated an ultra-performance liquid chromatography-tandem mass spectrometry method for the quantification of 15 common non-tricyclic antidepressants in serum: citalopram, desmethylcitalopram, desmethylfluoxetine, desmethylvenlafaxine, duloxetine, fluoxetine, fluvoxamine, melitracen, mianserin, mirtazapine, paroxetine, reboxetine, sertraline, trazodone and venlafaxine. The selected pharmaceuticals represent the most common non-tricyclic antidepressants available in Belgium. Serum samples (250 μL) were precipitated with methanol and acetonitrile containing 5 ng of the internal standards: mirtazapine-D 4 , paroxetine-D 5 , reboxetine-D 6 and venlafaxine-D 6 . The supernatant was evaporated at 56 °C under a flow of nitrogen and reconstituted in 100 μL of 50:50 methanol:water with 2 mM ammonium acetate. Five μL were injected and the target analytes were separated on a 100 × 2.1 mm ACQUITY BEH C18 column (Waters, Zellik, Belgium) using a Waters ACQUITY Ultra-Performance Liquid Chromatography system. The analytes were eluted within 7.5 min, using a gradient of 2 mM ammonium acetate and 0.1% formic acid in water and in methanol at a flow rate of 0.4 mL/min. Quantification was performed on a Waters triple-quadrupole ACQUITY TQD using multiple reaction monitoring (MRM) in positive mode (2 MRMs per analyte). A six-point calibration curve was used to cover a large concentration range from 10 ng/mL to 1000 ng/mL. The developed method was validated in terms of selectivity, linearity, precision, accuracy, matrix effect, recovery and stability. No interfering signals from matrix, internal standard or tricyclic antidepressant drugs were found. Limits of quantification and limits of detection ranged from 10 to 85 ng/mL and 2.0 to 28 ng/mL respectively. Ion suppression from matrix effects varied from 36 to 108% for the compounds and 47 to 102% for the internal standards. Process efficiency varied from 22 to 106%. Intra- and interassay precision varied from 87.7 to 96.5% and 87.7 to 96.9%, respectively. The bias of the assay was lower than 15% for all the compounds, except the lowest concentrations of desmethylcitalopram and trazodone (bias < 20%). Good validation performance was obtained thanks to the use of selected deuterated internal standards that adjusted for all of the compounds. This method is therefore suitable for quantification in suspected overdose cases and can be use in a clinical Toxicology lab in a daily basis.

A UPLC-MS Method for the Determination of Ofloxacin Concentrations in Aqueous Humor.

A rapid, simple, and specific method based on ultra performance liquid chromatography (UPLC) with mass spectrometry detection has been developed for quantitative analysis of ofloxacin in human aqueous humor using tobramycin as internal standard (IS). Chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 Shield column (150 × 2.1 mm, 1.7 μm) eluted with 95:5 water: acetonitrile (v/v) containing 0.1% formic acid and a flow rate of 0.3 mL/minute. The total analysis time was three minutes with ofloxacin eluting at 1.67 ± 0.03 minutes. The linearity of the method ranged from 0.1 to 8 μg/mL with r2 = 0.998. The method was validated according to FDA guidelines with respect to linearity, accuracy, precision, specificity, and stability. The limits of detection and quantification were 0.03 and 0.10 μg/mL, respectively. The developed method was successfully applied to the analysis of samples that have been obtained from patients.

Simultaneous Determination of Eight Bioactive Constituents in Shensong Yangxin Capsule by UPLC

ObjectiveTo develop a ultra performance liquid chromatography (UPLC) method for the quality evaluation of Shensong Yangxin Capsule (SYC). MethodsThe Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm) was used. Acetonitrile and water containing 0.1% phosphoric acid were used as mobile phases of UPLC with gradient elution. The detection wavelengths were set at 203 (ginsenoside Rb1), 286 (salvianolic acid B), 230 (paeoniflorin), 221 (schisantherin A), 280 (sodium danshensu), 327 (chlorognenic acid), 335 (spinosin), and 345 nm (berberine hydrochloride), respectively. The flow rate was set at 0.4 mL/min and column temperature was 35 °C. ResultsThe contents of paeoniflorin, salvianolic acid B, schisantherin A, sodium danshensu, chlorognenic acid, spinosin, berberine hydrochloride, and ginsenoside Rb1 were determined from 10 batches of SYC. ConclusionThe method of the quality evaluation of SYC has acceptable precision, reproducibility, and stability, which could be used as a new method for the quality control of SYC.