Development and certification of a reference material for zearalenone in maize germ oil.

Zearalenone (ZEN), an estrogenic mycotoxin produced by several species of Fusarium fungi, is a common contaminant of cereal-based food worldwide. Due to frequent occurrences associated with high levels of ZEN, maize oil is a particular source of exposure. Although a European maximum level for ZEN in maize oil exists according to Commission Regulation (EC) No. 1126/2007 along with a newly developed international standard method for analysis, certified reference materials (CRM) are still not available. To overcome this lack, the first CRM for the determination of ZEN in contaminated maize germ oil (ERM®-BC715) was developed in the frame of a European Reference Materials (ERM®) project according to the requirements of ISO Guide 35. The whole process of CRM development including preparation, homogeneity and stability studies, and value assignment is presented. The assignment of the certified mass fraction was based upon an in-house study using high-performance liquid chromatography isotope dilution tandem mass spectrometry. Simultaneously, to support the in-house certification study, an interlaboratory comparison study was conducted with 13 expert laboratories using different analytical methods. The certified mass fraction and expanded uncertainty (k = 2) of ERM®-BC715 (362 ± 22) µg kg-1 ZEN are traceable to the SI. This reference material is intended for analytical quality control and contributes to the improvement of consumer protection and food safety.

Citrinin Determination in Food and Food Supplements by LC-MS/MS: Development and Use of Reference Materials in an International Collaborative Study.

The development of incurred reference materials containing citrinin (CIT) and their successful application in a method validation study (MVS) in order to harmonize CIT determination in food and food supplements are demonstrated. CIT-contaminated materials made of red yeast rice (RYR), wheat flour, and Ginkgo biloba leaves (GBL), as well as food supplements made of red yeast rice (FS-RYR) and Ginkgo biloba leaves (FS-GBL), were manufactured in-house via fungal cultivation on collected raw materials. The homogeneity and stability from randomly selected containers were verified according to the ISO 13528. CIT was found to be homogenously distributed and stable in all contaminated materials, with no significant degradation during the timescale of the MVS when storage was performed up to +4 °C. Next, an MVS was organized with eighteen international laboratories using the provided standard operating procedure and 12 test materials, including three RYRs (blank, <50 µg/kg, <2000 µg/kg), two wheat flours (blank, <50 µg/kg), two GBL powders (blank, <50 µg/kg), three FS-RYRs (blank, <50 µg/kg, <2000 µg/kg), and two FS-GBLs (blank, <50 µg/kg). The results of seven CIT-incurred materials showed acceptable within-laboratory precision (RSDr) varying from 6.4% to 14.6% and between-laboratory precision (RSDR) varying from 10.2% to 37.3%. Evidenced by HorRat values < 2.0, the results of the collaborative trial demonstrated that the applied analytical method could be standardized. Furthermore, the appropriateness of producing CIT reference materials is an important step towards food and feed quality control systems and the organization of proficiency tests.

Mass Spectral Filtering by Mass-Remainder Analysis (MARA) at High Resolution and Its Application to Metabolite Profiling of Flavonoids.

Flavonoids represent an important class of secondary metabolites because of their potential health benefits and functions in plants. We propose a novel method for the comprehensive flavonoid filtering and screening based on direct infusion mass spectrometry (DIMS) analysis. The recently invented data mining procedure, the multi-step mass-remainder analysis (M-MARA) technique is applied for the effective mass spectral filtering of the peak rich spectra of natural herb extracts. In addition, our flavonoid-filtering algorithm facilitates the determination of the elemental composition. M-MARA flavonoid-filtering uses simple mathematical and logical operations and thus, it can easily be implemented in a regular spreadsheet software. A huge benefit of our method is the high speed and the low demand for computing power and memory that enables the real time application even for tandem mass spectrometric analysis. Our novel method was applied for the electrospray ionization (ESI) DIMS spectra of various herb extract, and the filtered mass spectral data were subjected to chemometrics analysis using principal component analysis (PCA).

The bioavailability and excretion of an antitussive compound IAsp-N-Glc in rats by validated UPLC-MS/MS methods.

IAsp-N-Glc is a potential antitussive agent that is first reported to be isolated from Ginkgo Semen, but the bioavailability and excretion of IAsp-N-Glc are unknown. Therefore, we carried out our study to obtain the bioavailability and excretion profiles of IAsp-N-Glc in rats. Rapid, specific, and reliable quantification methods for the measurement of IAsp-N-Glc in rat plasma and fecal samples by using ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry were developed and validated. A C18 column was used for the separation of IAsp-N-Glc and internal standards, and water (containing 0.1% formic acid) and acetonitrile were chosen as the mobile phase for the separation in the flow-gradient mode. In the ranges of 37.5-7500 ng/mL and 120-30000 ng/mL, the calibration curves of IAsp-N-Glc exhibited satisfactory linearity for plasma and fecal samples with each linear correlation coefficient higher than 0.99, respectively. The methods were reproducible and reliable. The analytes were stable, and no apparent matrix effects were observed. The bioanalytical methods were successfully used to study the pharmacokinetics and excretion of IAsp-N-Glc in rats. Oral administration of IAsp-N-Glc exhibited a low absolute oral bioavailability (1.83±0.09%), and 59.63±6.29% of IAsp-N-Glc was excreted in feces. This report is the first to describe the bioavailability and excretion of IAsp-N-Glc in rats and will lay the foundation for the in-depth study and drug development of IAsp-N-Glc.

Analysis of cobalt for human sports drug testing purposes using ICP- and LC-ICP-MS.

Due to the current demands in the fight against manipulation of blood and blood components, commonly referred to as "blood doping" in sports drug testing, specific and sensitive detection methods enabling the detection of prohibited substances and methods of doping are required. Similar to illicit blood transfusions, erythropoiesis stimulating agents have been shown to be misused in sport, aiming at improving an athlete's aerobic capacity and endurance performance. Amongst other strategies, the administration of ionic cobalt (Co2+ ) can increase the number of erythrocytes by stimulating the endogenous erythropoietin (EPO) biosynthesis. Conversely, several organic Co-containing compounds such as cyanocobalamin (vitamin B12) are not prohibited in sports, and thus, an analytical differentiation of permitted and banned contributions to urinary Co-concentrations is desirable. An excretion study with daily applications of either 1 mg of CoCl2 or 1 mg of cyanocobalamin was conducted with 20 volunteers over a period of 14 consecutive days. Urine, plasma, and concentrated red blood cells were analyzed for their cobalt content. The samples were collected starting 7 days before the administration until 7 days after. Total Co concentrations were analyzed by using inductively coupled plasma mass spectrometry (ICP-MS), which yielded significantly elevated levels exclusively after inorganic cobalt intake. Furthermore, a liquid chromatography (LC)-ICP-MS approach was established and employed for the simultaneous determination of organically bound and inorganic cobalt by chromatographic separation within one single run. The analytical approach offers the option to further develop detection methods of illegal Co2+ supplementation in sport.

Concurrent identification of 11 major primary active compounds in Huangqin Qingfei Decoction by liquid chromatography tandem mass spectrometry via liquid chromatography tandem mass spectrometry.

Huangqin Qinfei Decoction (HQD) is a traditional Chinese medicine that is administered for acute pneumonia, bronchial inflammation, acute bronchitis and acute lung infection. In this study, we used liquid chromatography linked with tandem mass spectrometry (LC-MS/MS) for the concurrent identification of 11 bioactive compounds; namely, baicalin, baicalein, wogonoside, scutellarin, wogonin, oroxylin A, geniposide, genipin, geniposidic acid, chlorogenic acid, and crocin-I, for the quality control of HQD. The evaluation was conducted on an Agilent Poroshell 120 EC-C18 (2.1mm×100mm, 2.7µm) with gradient elution in the mobile phase with 0.1% formic acid and 1mM/L ammonium acetate in water as solvent A and methanol as solvent B at a flow rate of 0.3mL/min in under 12 min. Mass spectrometric detection was conducted in the selected reaction monitoring mode utilizing electro spray ionization in the positive and negative modes. Every one of the calibration curves had good linearity with R2 >0.9992. Intra-day and inter-day accuracies for every one of the evaluated components were expressed as the relative standard deviation (RSD) from 1.72%-5.02% and 0.63%-5.99%, respectively. The recuperation of the 11 compounds that were measured at the three concentrations was within 94.05%-105.18%, with the RSD ≤ 6.26%. The use of this method was determined through the effective evaluation of 11 compounds in 5 batches of HQD. The confirmed method is precise, sensitive, and effective for identifying the contents of the chosen compounds in HQD for quality control.

Separation of betacyanins from Iresine herbstii Hook. ex Lindl. leaves by high-speed countercurrent chromatography in a polar solvent system.

Betacyanins, natural plant pigments, from Iresine herbstii Hook. ex Lindl. leaf extract were separated for the first time by high-speed countercurrent chromatography (HSCCC) in a highly polar solvent system composed of PrOH-ACN-(NH4)2SO4satd.soln-H2O (1.0:0.5:1.2:1.0; v/v/v/v) in the tail-to-head mode. The flow rate of the mobile phase was 2.0 ml/min and the column rotation speed was 860 rpm. The retention of the stationary phase was 81.0%. For the identification of separated betacyanins in the crude extract and in the HSCCC fractions, as well as for the molecular formulas and multi-step fragmentation pattern elucidation, liquid chromatography with tandem mass spectrometry and high-resolution ion-trap time-of-flight mass spectrometry were performed. The innovative application of the HSCCC system for fractionation of the betacyanins present in I. herbstii leaves enabled effective separation as well as preconcentration of the pigments for further low- and high-resolution LC-MS/MS analysis. HSCCC separation enabled identification of 22 betacyanins, of which 18 had not been detected previously in the leaves of I. herbstii, and four of these betacyanins (sinapoyl-gomphrenin and coumaroyl-gomphrenin as well as their epimers) were identified for the first time in the Iresine genus.

Collaborative study: Quantification of total folate in food using an efficient single-enzyme extraction combined with LC-MS/MS.

Quantification of the specific folate vitamers to estimate total folate in foods is not standardized. A collaborative study, including eight European laboratories, was conducted in order to determine the repeatability and reproducibility of the method for folate quantification in foods using the plant-origin γ-glutamyl hydrolase as part of the extraction procedure. The seven food samples analyzed represent the food groups; fruits, vegetables, dairy products, legumes, offal, fish, and fortified infant formula. The homogenization step was included, and six folate vitamers were analyzed using LC-MS/MS. Total folate content, expressed as folic acid equivalent, was 17-490 µg/100 g in all samples. Horwitz ratio values were within the acceptable range (0.60-1.94), except for fish. The results for fortified infant formula, a certified reference material (NIST 1869), confirmed the trueness of the method. The collaborative study is part of a standardization project within the Nordic Committee on Food Analysis (NMKL).

An oligosaccharide-marker approach to quantify specific polysaccharides in herbal formula by LC-qTOF-MS: Danggui Buxue Tang, a case study.

Polysaccharides have broad bioactivities and are major components of water decoction of herb formulae. However, the quality control of polysaccharides remains a challenge. Oligosaccharide-fragment approach has been considered in elucidating chemical structures of polysaccharides, but never been used for quantitation. Using reference chemicals and a real sample Danggui Buxue Tang (DBT) in this study, an oligosaccharide-marker approach was established to quantify specific polysaccharides. Firstly, linear relationships between parent polysaccharides and hydrolysis-produced daughter oligosaccharides were verified using reference polysaccharides. Then in case of DBT, two fluorescence-labeled oligosaccharides with high specificity to individual parent polysaccharides were selected as markers. They were easily isolated and identified. Their potential in quantification of parent polysaccharides were satisfactorily validated in terms of linearity (r≥0.99), repeatability (RSD ≤ 8.4 %), and spike recovery (≥80 %). This method could be a promising approach for quality assessment of polysaccharides in herbal formulae.

Profiling and identification of aqueous extract of Cordyceps sinensis by ultra-high performance liquid chromatography tandem quadrupole-orbitrap mass spectrometry.

Characterization of aqueous extract in traditional Chinese medicine (TCM) is challenging due to the poor retention of the analytes on conventional C18 columns. This study presents a systematic characterization method based on a rapid chromatographic separation (8 min) on a polar-modified C18 (Waters Cortecs T3) column of aqueous extract of Cordyceps sinensis. UHPLC-HRMS method was used to profile components in both untargeted and targeted manners by full MS/PIL/dd-MS2 acquisition approach. The components were identified or tentatively identified by reference standards comparison, fragmentation rules elucidation and available databases search. A total of 91 components, including 10 nucleobases, 20 nucleosides, 39 dipeptides, 18 amino acids and derivatives and 4 other components, were characterized from the aqueous extract of C. sinensis. And this was the first time to systematically report the presence of nucleosides and dipeptides in C. sinensis, especially for modified nucleosides. The chemical basis inquiry of this work would be beneficial to mechanism exploration and quality control of C. sinensis and related products. Meanwhile, this work also provided an effective solution for characterization of aqueous extract in TCM.

Modified QuEChERS purification and Fe3O4 nanoparticle decoloration for robust analysis of 14 heterocyclic aromatic amines and acrylamide in coffee products using UHPLC-MS/MS.

Based on QuEChERS dispersed purification, Fe3O4 nanoparticle decoloration and UHPLC-MS/MS, a robust and sensitive method was established for simultaneous analysis of 14 heterocyclic aromatic amines (HAAs) and acrylamide (AA) in coffee products. Sample was extracted by 90% acetonitrile water (v/v), dispersed with primary secondary amine (PSA) and further purified with Fe3O4 nanoparticle. Then, 15 analytes were detected using ESI positive ion under MRM mode. Good linearity was observed for all analytes in the range of 0.2-100 µg/L with the determination coefficients being above 0.996. Limits of detection (S/N ≥ 3) and limits of quantification (S/N ≥ 10) were in the range of 0.02-0.15 µg/L and 0.2-0.7 µg/L, respectively. The intra-day average recoveries were between 81.6% and 100%, and the intra-day precisions ranged from 4.3% to 9.0%. The inter-day average recoveries were in the range of 81.0-101% with precisions ranging from 5.0% to 7.8%. Results indicated that the combination of PSA and Fe3O4 exhibited superior purification and adsorption effects for removing pigments and acid compounds. Real samples analysis indicated that coffee products were widely contaminated with AA, harman and norharman.

External Quality Assessment of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) assays.

The discovery that mutations of the CYP24A1 gene are a cause of idiopathic infantile hypercalcemia (IIH) has revived interest in measuring serum 24,25(OH)2D3. Several studies have also suggested that a high 25-hydroxyvitamin D3(25-OHD3):24,25(OH)2D3 ratio might provide additional diagnostic information in the investigation of vitamin D deficiency. Measurement of 24,25(OH)2D3 is necessarily restricted to laboratories with mass spectrometry methods although cross reactivity of the metabolite in immunoassays for 25-OHD is a potential cause of misleading results. The international External Quality Assessment (EQA) scheme for vitamin D metabolites (DEQAS) was set up in 1989. In 2013 DEQAS became an accuracy based EQA for 25-OHD with 'target values' assigned by the National Institute of Standards and Technology (NIST) Reference Measurement Procedure (RMP). A pilot scheme for serum 24,25(OH)2D3 was started in 2015 and participants were asked to measure the metabolite on each of the 5 samples sent out for 25-OHD. Inter-laboratory agreement was poor but this may reflect methodological differences, in particular different approaches to assay standardization. An important potential contribution to reducing variability among assays was the development by NIST of a 24,25(OH)2D3 RMP and its use in assigning values to SRMs 972a, 2973 and 2971, supported by the NIH Office of Dietary Supplements (ODS) as part of the Vitamin D Standardization Program (VDSP) effort.

An integrative UHPLC-MS/MS untargeted metabonomics combined with quantitative analysis of the therapeutic mechanism of Si-Ni-San.

A UHPLC-MS/MS untargeted serum metabonomic method combined with quantitative analysis of five potential biomarkers in rat serum was developed and validated, to further understand the anti-liver injury effect of Si-Ni-San and its mechanism on liver injury rats in this study. The metabolites were separated and identified on BEH C18 column (100 mm × 2.1 mm, 1.7 µm) using the ACQUITY UHPLC-MS system (Waters Corp., Milford, MA, USA). Principal component analysis (PCA) was used to identify potential biomarkers. Primary potential biomarkers including phenylalanine, tryptophan, Glycochenodeoxycholic acid (GCDCA) and hysophosphatidylcholine (LPC), which were related to amino acid metabolism, lipid metabolism, bile acid biosynthesis and oxidation-antioxidation balance, were found in the untargeted metabonomic research. Moreover, these targeted biomarkers were further separated and quantified in multiple-reaction monitoring (MRM) with positive ionization mode. The proposed method was linear for each analyte with correlation coefficients over 0.99. The intra- and inter-day precision values (relative standard deviation, RSD) were less than 13.1% and accuracy (relative error, RE) was from -9.5% to 10.3% at all quality control (QC) levels. The validated method was successfully applied to study the serum samples of control group, model group, positive control group (silymarin group) and Si-Ni-San group in rats.

Multiclass mycotoxins in lotus seeds analysed by an isotope-labelled internal standard-based UPLC-MS/MS.

OBJECTIVES: This study aimed to explore the residue levels of multiclass mycotoxins in medicinal and edible lotus seeds. METHODS: A rapid and reliable isotope-labelled internal standard-based UPLC-MS/MS method was developed and validated for sensitive and accurate analysis of multiclass mycotoxins including aflatoxins (AFB1 , AFB2 , AFG1 and AFG2 ), ochratoxin A (OTA), zearalenone (ZEN), deoxynivalenol (DON), fumonisins (FB1 and FB2 ), T-2 and HT-2 toxins in lotus seeds. Some critical conditions such as extract solution with the addition of isotope-labelled internal standard, type of mobile phase and the elution condition were scientifically optimized. The 11 mycotoxins obtained satisfactory resolution and sensitive detection in multiple reactions monitoring scanning mode combined with the ion switching technology in positive and negative ion switching mode. KEY FINDINGS: The developed isotope-labelled internal standard-based UPLC-MS/MS method exhibited an approving linearity (r ≥ 0.9984), high sensitivity (limit of detection in the range of 0.015-30.05 µg/kg), acceptable precision (RSDs ≤6.3%) and good recovery (76.0-116.0%) for 11 analytes, respectively. Ten batches of real lotus seed samples were tested, and three batches out of which were contaminated with AFB1 , FB2 , T-2 and ZEN. AFB1 showed the highest occurrence rate (30%) with contents of 10.50 and 8.32 µg/kg in two samples over the official limit (5.0 µg/kg). CONCLUSIONS: The monitoring of multiclass mycotoxins in Chinese herbal medicines is in great urgency to ensure the security of consumers. The proposed method could be further utilized for simple, sensitive and rapid detection of more mycotoxins in other complex matrices to compensate for matrix effects.

Compensation for matrix effects in GC analysis of pesticides by using cucumber extract.

Matrix effects (MEs) can adversely affect quantification in pesticide residue analysis using GC. Analyte protectants (APs) can effectively interact with and mask active sites in the GC system, and are added individually or in combination to sample extracts and calibration solutions to minimize errors related to MEs. Unfortunately, APs cannot sufficiently compensate for MEs in all cases. Plant extracts, containing a broad range of natural compounds with AP properties, can also be used for this purpose. In this study, the applicability of cucumber extract as a natural AP mixture was investigated both alone and in combination with traditional APs. Extracts of two selected difficult matrices (onion and garlic) were prepared according to the citrate-buffered QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure. ME values of 40 representative GC-amenable pesticides were compared when calibrating against standards in pure solvent and in cucumber extract, with and without the addition of APs. Using a GC system with a contaminated inlet liner, the use of a cucumber-based calibration solution decreased MEs remarkably. The combination of APs with cucumber raw extract further decreased MEs, resulting in more than 85% of the tested pesticides showing ≤ 10% ME in onion and ≤ 20% ME in garlic. These results demonstrate that the preparation of calibration standards based on cucumber extracts (with or without the addition of APs) is a very useful and practical approach to compensate for MEs in pesticide residue analysis using QuEChERS and GC-MS/MS. The use of various internal standards is furthermore critically discussed.

Development and validation of a UFLC-MS/MS method for simultaneous quantification of sixty-six saponins and their six aglycones: Application to comparative analysis of red ginseng and white ginseng.

A new and sensitive ultra fast liquid chromatography coupled with electrospray ionization triple quadrupole tandem mass spectrometry (UFLC-MS/MS) method was developed to evaluate the quality of Red ginseng (RG) and to find out its chemical markers by comparing with multi-batches of RG and white ginseng (WG). This innovative method could quantify sixty-six saponins and their six aglycones including 10 pairs of 20(S) and 20(R) epimers within 35 min simultaneously. All compounds could be determined in individual multiple-reaction monitoring channel without interference, and the optimized method was rapid, accurate, precise, reproducible and efficient. Using the orthogonal partial least squared discriminant analysis, ginsenosides Rg5, Rh4, Rk1, Rs4, F4, and 20(S)-Rg3 were found to be the characteristic components of RG, the six compounds should be suggested as quality control markers to distinguish RG from WG. These findings will be significant for standardizing the processing procedures of RG and ensuring the consistent quality, as well as consequently the efficacy of RG in clinical applications. Results will be helpful in providing crucial chemical profiles of RG.

Vitamin D Assays.

The number of requests for vitamin D metabolite measurements has increased dramatically over the past decade leading commercial laboratories to develop rapid high throughput assays. The measurement of 25-hydroxyvitamin D (25[OH]D) and to a lesser extent 1,25-dihydroxyvitamin D (1,25[OH]2D) dominates these requests, but requests for multiple metabolite measurements in the same sample are also increasing. The most commonly used methods include immunoassays and liquid chromatography/mass spectrometry (LC-MS). Each method has its advantages and disadvantages, but with improvements in technology, especially in LC-MS, this method is gaining ascendance due to its greater precision and flexibility. The use of standards from the National Institutes of Standards and Technology has substantially reduced the variability from laboratory to laboratory, thereby improving the reliability of these measurements. Although the current demand is for measurement of total vitamin D metabolite levels, these metabolites circulate in blood tightly bound to vitamin D binding protein (DBP) and albumin with less than 1% free. The free concentration may be a more accurate indicator of vitamin D status especially in individuals with DBP levels that deviate from the normal population. Thus, methods to measure the free concentration at least of 25(OH)D are becoming available and may supplement if not replace measurements of total levels.

Use of UHPLC-TripleQ with synthetic standards to profile anti-inflammatory hydroxycinnamic acid amides in root barks and leaves of Lycium barbarum.

Hydroxycinnamic acid amides (HCAA) are the secondary metabolites ubiquitously exist in flowering plants, formed by condensation between hydroxycinnamates and mono or polyamines. HCAA species not only serve multiple functions in plant growth and development, but also exert significant positive effects on human health. In this study, we combined organic synthesis and UPHLC-TripleQ-MS/MS specifically targeting at HCAA species. The method was fully validated with respect to specificity, linearity, intra- and inter-day precision and accuracy, limit of detection (LOD), limit of quantification (LOQ), recovery, and reproducibility. We applied this method to identify and quantify HCAAs from the root barks and leaves of Lycium barbarum. HCAA species were reported in leaves for the first time, and 10 new HCAA species were further identified in root barks in addition to the ones reported in the literature. We also examine anti-inflammatory properties of identified HCAAs species. Seven HCAA compounds had a potent NO inhibitory effect with IC50 as low as 2.381 µM (trans-N-caffeoyl phenethylamine). Our developed method largely improved analytical sensitivity of HCAAs species that potentially contributes to plant metabolomics studies.

Development and validation of an UHPLC-MS/MS approach for simultaneous quantification of five bioactive saponins in rat plasma: Application to a comparative pharmacokinetic study of aqueous extracts of raw and salt-processed Achyranthes bidentata.

A simple, accurate and sensitive ultra high-performance liquid chromatography-tandem mass spectrometry approach was established for the simultaneous determination of ß-ecdysterone, 25S-inokosterone, ginsenoside Ro, chikusetsusaponin IV and chikusetsusaponin IVa in rat plasma after oral administration of raw and salt-processed Achyranthes bidentata extract. The saponins were completely separated on a Waters BEH C18 UHPLC column by using acetonitrile/0.1% formic acid-water as mobile phases. The mass analysis was performed in a triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) with negative scan mode. The sample preparations for protein removal were accomplished using a simple acetonitrile precipitation method. The calibration curves displayed good linearity (r2 > 0.9998) with the concentration ranges of 24.4-6100 ng mL-1, 25.6-6400 ng mL-1, 20.4-8500 ng mL-1, 21.6-5400 ng mL-1, 21.6-6100 ng mL-1 for the five saponins, respectively. The intra-day and inter-day precisions (RSD) of the five saponins were less than 3.95% and the bias of the accuracies ranged from -4.50% to 4.84%. The extraction recoveries of the five saponins ranged from 95.2% to 104.8% and the matrix effects were satisfactory. In comparison with the raw group, the parameters of Cmax and AUC0-t of ß-ecdysterone, 25S-inokosterone, ginsenoside Ro, and chikusetsusaponin IVa elevated remarkably (p < 0.05) after oral delivery of the extract of salt-processed Achyranthes bidentata, which revealed that salt-processing could increase bioavailability of ß-ecdysterone, 25S-inokosterone, ginsenoside Ro and chikusetsusaponin IVa.

Simultaneous quantification of four antiretroviral drugs in breast milk samples from HIV-positive women by an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method.

The primary strategy to avoid mother-to-child transmission of human immunodeficiency virus (HIV) through breastfeeding is administration of highly active antiretroviral therapy (HAART) to HIV-positive pregnant women. Because significant changes in the pharmacokinetics of antiretroviral (ARV) drugs occur during pregnancy, quantifying HAART and the viral load in breast milk in this population is essential. Here, we developed an analytical assay for the simultaneous quantification of four ARV drugs in breast milk using ultra-performance liquid chromatography coupled to tandem mass spectrometry. We validated this method following Mexican and international guidelines. ARV drugs. We extracted the ARV drugs from 200 µL samples of breast milk and detected these drugs in a triple quadrupole mass spectrometer with positive electrospray ionization. The validated concentration ranges (ng/mL) for zidovudine, lamivudine, lopinavir, and ritonavir were 12.5-750, 50-2500, 100-5000 and 5 to 250, respectively. Additionally, the absolute recovery percentages (and matrix effects) were 91.4 (8.39), 88.78 (28.75), 91.38 (11.77) and 89.78 (12.37), respectively. We determined that ARV drugs are stable for 24 h at 8°C and 24°C for 15 days at -80°C. This methodology had the capacity for simultaneous detection; separation; and accurate, precise quantification of ARV drugs in human breast milk samples according to Mexican standard laws and United States Food and Drug Administration guidelines.