High-performance Liquid Chromatographic Assay Research Articles

Diminishing oocyte quality with advancing age is associated with deficiency of nitric oxide synthase cofactors, tetrahydrobiopterin, and zinc, in mouse oocytes

To study the implications of decreased zinc and tetrahydrobiopterin (H4B) associated with chronological aging on oocyte quality using a mouse model. H4B and zinc are essential cofactors for nitric oxide synthase (NOS), because they aid in electron transfer and dimeric stability, and their bioavailability is crucial in regulating NOS coupling. We have previously shown that sufficient levels of nitric oxide (NO) are essential for maintaining oocyte quality and activation, and NO levels decrease in the oocyte as a function of age. Thus, it is plausible that zinc and H4B may decrease as a function of age, resulting in NOS dysfunction with subsequent depletion of NO. Additionally, increased production of reactive oxygen species from the monomeric form can further disrupt oocyte quality and NO bioavailability. Experimental laboratory study. Laboratory. B6D2F1 mice. Sibling oocytes were retrieved from super-ovulated B6D2F1 mice from 3 age groups: 8-14 weeks (young breeders [YBs]), 48-52 weeks (retired breeders [RBs]), and 80-84 weeks (old animals [OAs]). Oocytes were scored for ooplasmic/spindle microtubule (MT) morphology, chromosomal alignment, and cortical granule (CG) intactness using immunofluorescence and confocal microscopy with 3 dimension image reconstruction and subjected to an high-performance liquid chromatography assay to measure the concentrations of H4B and its metabolites, as well as the zinc measurement using atomic absorption spectrophotometry. Oocyte scoring showed a reduction in "good" quality oocyte percentage as age increases, with YB having the highest percentage of quality oocytes followed by RB and OA. The high-performance liquid chromatography analysis showed a significant progressive decrease in total H4B in RB and OA (0.00098 picogram (pg)/oocyte and 0.00069 pg/oocyte, respectively) compared with YB (0.00125 pg/oocyte). Atomic absorbance spectrophotometry revealed a significant progressive decrease in zinc concentration in RB and OA compared with YB (8.45 pg/oocyte and 5.82 pg/oocyte vs. 10.05 pg/oocyte, respectively). Age-related diminution in oocyte quality is paralleled by a decline in the levels of H4B and zinc. The resultant deficiency in the oocytes can lead to the inability of NOS to maintain dimerization. Consequent uncoupling of NOS generates superoxide instead of NO, which participates in a multitude of reactions contributing to oxidative stress. Therefore, dysfunction of NOS secondary to zinc and H4B loss is a major mechanism involved in reactive oxygen species generation and oocyte quality deterioration related to the chronological age.

Sustained Release of Antifungal and Antibacterial Agents from Novel Hybrid Degradable Nanofibers for the Treatment of Polymicrobial Osteomyelitis

This study aimed to develop a drug delivery system with hybrid biodegradable antifungal and antibacterial agents incorporated into poly lactic-co-glycolic acid (PLGA) nanofibers, facilitating an extended release of fluconazole, vancomycin, and ceftazidime to treat polymicrobial osteomyelitis. The nanofibers were assessed using scanning electron microscopy, tensile testing, water contact angle analysis, differential scanning calorimetry, and Fourier-transform infrared spectroscopy. The in vitro release of the antimicrobial agents was assessed using an elution method and a high-performance liquid chromatography assay. The in vivo elution pattern of nanofibrous mats was assessed using a rat femoral model. The experimental results demonstrated that the antimicrobial agent-loaded nanofibers released high levels of fluconazole, vancomycin, and ceftazidime for 30 and 56 days in vitro and in vivo, respectively. Histological assays revealed no notable tissue inflammation. Therefore, hybrid biodegradable PLGA nanofibers with a sustainable release of antifungal and antibacterial agents may be employed for the treatment of polymicrobial osteomyelitis.

Rapid and Simultaneous Detection of Aflatoxin B1, Zearalenone, and T-2 Toxin in Medicinal and Edible Food Using Gold Immunochromatographic Test Strip.

(1) Background: Medicinal and edible food and traditional Chinese medicine have been used to treat various diseases. However, their safety has not been thoroughly assessed. (2) Methods: An immunochromatographic test strip (ICS) was used for the first time to screen some mycotoxins, including aflatoxin B1 (AFB1), zearalenone (ZEN), and T-2 toxin, in medicinal and edible food and traditional Chinese medicine. Antibody/nano-gold particle coupling was used with the prepared ICS, and the pH, monoclonal antibody concentration, and antigen amount were optimized. The extraction sample solution was diluted 10 times with phosphate-buffered saline containing 0.5% Tween-20 and 0.05% sodium dodecyl sulfate to remove the complex matrix in medicinal and edible food. (3) Results: Under optimal conditions, the sensitivities of the developed ICS for AFB1, ZEN, and T-2 were 0.5, 5.0, and 5.0 ng/mL, respectively. Among the 30 medicinal and edible food samples tested, two samples (both of sand jujube kernels) were positive, and the results were verified by high-performance liquid chromatography and enzyme-linked immunosorbent assay and were consistent with the ICS test results. (4) Conclusions: The ICS could be used for rapid screening and simultaneous detection of mycotoxins at medicinal and edible food storage facilities.

Prevalence of Poor Quality Ciprofloxacin and Metronidazole Tablets in Three Cities in Cameroon.

Quality is one of the essential components of medicines and needs to be ensured to preserve the population's health. This can be achieved through post-marketing quality control of medicines and is one of the most important duties of national regulatory authorities. In collaboration with the Cameroonian National Drug Quality Control and Valuation Laboratory, the decision was made to initiate a prevalence study to assess the quality of antiinfective medicines in Cameroon. A total of 150 samples of ciprofloxacin tablets and 142 samples of metronidazole tablets were collected from 76 licensed pharmacies and 75 informal vendors in three cities in Cameroon using a random strategy wherever possible and a mystery shopper approach. Three tests were carried out on each of the samples. Visual inspection allowed to find two falsified samples (0.7%) due to lack of information about the manufacturing company, and five more samples (1.7%) were deemed to be substandard due to flaws in the product. An additional 13 samples (4.5%) failed disintegration testing, and six (2.1%) others failed high-performance liquid chromatography assay testing due to insufficient active pharmaceutical ingredient (API) content. All samples were found to contain some API. A prevalence of 7.9% substandard or falsified (SF) medicines was found. Moreover, the prevalence of outlets selling SF medicines was greater in the informal sector (26.7%) than in the formal sector (2.6%). Although the prevalence of SF medicines found was low, efforts need to be made by national regulatory authorities to monitor the pharmaceutical market more closely.

Antibody enhanced HPLC for serotype-specific quantitation of polysaccharides in pneumococcal conjugate vaccine

Bacterial infection remains as one of the major healthcare issues, despite significant scientific and medical progress in this field. Infection by Streptococcus Pneumoniae (S. Pneumoniae) can cause pneumonia and other serious infectious diseases, such as bacteremia, sinusitis and meningitis. The pneumococcal capsular polysaccharides (CPS) that constitute the outermost layer of the bacterial cell are the main immunogens and protect the pathogen from host defense mechanisms. Over 90 pneumococcal CPS serotypes have been identified, among which more than 30 can cause invasive pneumococcal diseases that could lead to morbidity and mortality. Multivalent pneumococcal vaccines have been developed to prevent diseases caused by S. Pneumoniae. These vaccines employ either purified pneumococcal CPSs or protein conjugates of these CPSs to generate antigen-specific immune responses for patient protection. Serotype-specific quantitation of these polysaccharides (Ps) antigen species are required for vaccine clinical dosage, product release and quality control. Herein, we have developed an antibody-enhanced high-performance liquid chromatography (HPLC) assay for serotype-specific quantitation of the polysaccharide contents in multivalent pneumococcal conjugate vaccines (PCVs). A fluorescence-labeled multiplex assay format has also been developed. This work laid the foundation for a serotype-specific antigen assay format that could play an important role for future vaccine research and development.

Hydroxytyrosol Reduces Foam Cell Formation and Endothelial Inflammation Regulating the PPARγ/LXRα/ABCA1 Pathway.

Cholesterol accumulation in macrophages leads to the formation of foam cells and increases the risk of developing atherosclerosis. We have verified whether hydroxytyrosol (HT), a phenolic compound with anti-inflammatory and antioxidant properties, can reduce the cholesterol build up in THP-1 macrophage-derived foam cells. We have also investigated the potential mechanisms. Oil Red O staining and high-performance liquid chromatography (HPLC) assays were utilized to detect cellular lipid accumulation and cholesterol content, respectively, in THP-1 macrophages foam cells treated with HT. The impact of HT on cholesterol metabolism-related molecules (SR-A1, CD36, LOX-1, ABCA1, ABCG1, PPARγ and LRX-α) in foam cells was assessed using real-time PCR (RT-qPCR) and Western blot analyses. Finally, the effect of HT on the adhesion of THP-1 monocytes to human vascular endothelial cells (HUVEC) was analyzed to study endothelial activation. We found that HT activates the PPARγ/LXRα pathway to upregulate ABCA1 expression, reducing cholesterol accumulation in foam cells. Moreover, HT significantly inhibited monocyte adhesion and reduced the levels of adhesion factors (ICAM-1 and VCAM-1) and pro-inflammatory factors (IL-6 and TNF-α) in LPS-induced endothelial cells. Taken together, our findings suggest that HT, with its ability to interfere with the import and export of cholesterol, could represent a new therapeutic strategy for the treatment of atherosclerotic disease.

Evaluation the food safety of cultured fat via detection of residues of adipogenic differentiation cocktail in cultured fat with high performance liquid chromatography and enzyme-linked immunosorbent assay

Cultured fat is inducing adipose progenitor cells (APCs) to differentiate into mature adipocytes for consumption. The traditional adipogenic differentiation cocktail, including insulin, dexamethasone, indomethacin, isobutylmethylxanthine and rosiglitazone, has potential food safety problems in cultured fat. Therefore, the detection of these residues is necessary to ensure food safety. In this research, a method of high performance liquid chromatography (HPLC) was established to quantitatively analyze the potential residual content of dexamethasone, indomethacin, isobutylmethylxanthine and rosiglitazone in cultured fat and medium. Quantitative analysis showed that the content of four residues in cultured fat decreased to zero on Day 10. Subsequently, enzyme-linked immunosorbent assay (ELISA) was performed to detect the insulin content in the cultured fat and found that the insulin content in the cultured fat on Day 10 was 2.78 ± 0.21 μg/kg. After soaking with phosphate buffered saline (PBS), the insulin content decreased to 1.88 ± 0.54 μg/kg. In conclusion, this research provided an effective approach to clarify the content of potential residual components in cultured fat and it will provide reference for the safety of cultured fat in the future.

QUANTITATIVE GENETIC ANALYSIS OF PHENOLIC ACIDS IN OILSEED RAPE MEAL

Rapeseed meal, a by-product of oilseed extraction related to the agri-food and biofuel industries due to its favourable composition of essential amino acids, is currently utilised for animal feed. In this study, 166 doubled haploid (DH) rapeseed lines from the segregating Brassica napus doubled haploid population YE2-DH were genetically and chemically analysed for phenolic acids. The relationship between seed colour and phenolic acid fractions in B. napus was investigated using these analyses to improve the quality of rapeseed meal. High-performance liquid chromatography assays were used to estimate phenolic acid content, and the outcomes were used to identify quantitative trait loci (QTL). Nine quantitative feature loci for three distinct phenolic acid compounds were mapped to seven linkage groups. A minor QTL for sinapine was located on linkage group C05 in the same interval as the QTL for seed colour. On chromosome A09, two loci for phenolic acids colocalised with the main QTL for seed colour. Closely linked molecular markers for the target traits (seed colour, phenolic acids) identified during this study could be useful tools for breeding new oilseed rape cultivars with lower levels of antinutritive compounds.

Montane Rattlesnakes in México: Venoms of Crotalus tancitarensis and Related Species within the Crotalus intermedius Group.

The Crotalus intermedius group is a clade of rattlesnakes consisting of several species adapted to a high elevation habitat, primarily in México. Crotalus tancitarensis was previously classified as C. intermedius, until individuals occurring on Cerro Tancítaro in Michoacán, México, were reevaluated and classified as a new species (C. tancitarensis) based on scale pattern and geographic location. This study aimed to characterize the venom of C. tancitarensis and compare the venom profile to those of other species within the Crotalus intermedius group using gel electrophoresis, biochemical assays, reverse-phase high performance liquid chromatography, mass spectrometry, and lethal toxicity (LD50) assays. Results show that the venom profiles of species within the Crotalus intermedius group are similar, but with distinct differences in phospholipase A2 (PLA2), metalloproteinase PI (SVMP PI), and kallikrein-like serine proteinase (SVSP) activity and relative abundance. Proteomic analysis indicated that the highland forms produce venoms with 50-60 protein isoforms and a composition typical of type I rattlesnake venoms (abundant SVMPs, lack of presynaptic PLA2-based neurotoxins), as well as a diversity of typical Crotalus venom components such as serine proteinases, PLA2s, C-type lectins, and less abundant toxins (LAAOs, CRiSPs, etc.). The overall venom profile of C. tancitarensis appears most similar to C. transversus, which is consistent with a previous mitochondrial DNA analysis of the Crotalus intermedius group. These rattlesnakes of the Mexican highlands represent a radiation of high elevation specialists, and in spite of divergence of species in these Sky Island habitats, venom composition of species analyzed here has remained relatively conserved. The majority of protein family isoforms are conserved in all members of the clade, and as seen in other more broadly distributed rattlesnake species, differences in their venoms are largely due to relative concentrations of specific components.

Dicamba Degradation by Fenton-Like Process Using Iron/Biochar Composites

In this work, an iron/biochar composite was evaluated as a catalyst for 3,6-dichloro2-methoxybenzoic acid (dicamba) herbicide degradation by heterogeneous Fenton-like process. The biochar was produced from pyrolysis of coffee husks. After FeII adsorption by biochar, the material was submitted to chemical reduction, via borohydride, producing zero-valent iron nanoparticles (BIO-Fe0 ). The material was characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy and energy dispersive X-ray spectroscopy, Raman scattering spectroscopy, nitrogen adsorption/desorption analysis, among other techniques. The BIO-Fe0 is a heterogeneous material, and the main constituent elements are carbon (C), oxygen (O), silicon (Si), sulfur (S) and iron (Fe). The dicamba degradation was monitored by high-performance liquid chromatography and biological assays using beans (Phaseolus vulgaris) as indicator species. The commercial dicamba (500 mL; 250 mg L-1) was submitted to Fenton-like process (initial pH 3.20; BIO-Fe0 : 3 or 5 g; 1.00 mL H2O2 at 3.00 mmol L-1; 1.00 mL Al2(SO4)3 at 1.00 mol L-1; 25 °C and 5 h of reaction), presenting ca. 100% of degradation. These treated solutions were used in a biological assay. The intoxication levels were less 30%. These results show that BIO-Fe0 has the potential to be used in the dicamba degradation by Fenton-like processes.

All-in-one microfluidic device with an integrated porous filtration membrane for on-site detection of multiple salivary biomarkers

A hand-held microfluidic device is developed with an integrated filtration membrane for electrochemical monitoring of multiple salivary biomarkers. The platform consists of array of screen-printed electrodes, integrated with a high throughput microfluidic system that was developed via clean-room free 3D-printing technique. A ‘channel-inside-channel’ configuration is introduced to facilitate pump-less passive flow of saliva at the broad sensing chambers. In order to eliminate the salivary protein based biofouling problems, a flexible porous filtration membrane is integrated at the microfluidic inlet that avoids the passage of bulkier protein constituents. As the presence of separate insulator layer on the sensing electrodes adversely affects its integration with the fluidic channel, the electrode substrate itself is utilized as the insulator by exposing the bottom region of the printed electrodes as active region. Enzyme based electrochemical sensors are developed with suitable nanomaterials for monitoring glucose, lactate, cholesterol and uric acid from collected saliva samples. The device showed distinct differences in salivary glucose levels for healthy subjects before and after consuming glucose-rich drinks. The glucose sensor responses are validated with high performance liquid chromatography and commercial colorimetric assay methods, and observed good correlation with parallel finger prick blood glucose measurements with an average Pearson coefficient of 0.9161, demonstrating its efficacy in saliva based point-of-care analysis.

Hyperglycemia-associated Alzheimer's-like symptoms and other behavioral effects attenuated by Plumeria obtusa L. Extract in alloxan-induced diabetic rats.

Diabetes mellitus is a chronic metabolic complaint with numerous short- and long-term complications that harm a person's physical and psychological health. Plumeria obtusa L. is a traditional medicine used in the treatment of diabetes to reduce complications related to behavior. Plumeria is a genus with antipsychotic activities. The objective of this study was to examine the effects of a methanolic extract of Plumeria obtusa L. in the attenuation of diabetes, on symptoms of Alzheimer disease, and on other associated behavioral aspects. A single dose of alloxan was administered to an experimental group of rats to induce development of diabetes (150mg/kg, intraperitoneal) and the rats were then administered selected doses of methanolic extract of Plumeria obtusa L. (Po.Cr) or glibenclamide (0.6mg/kg) for 45 consecutive days. Behavioral effects were evaluated using three validated assays of anxiety-related behavior: the open field test, the light and dark test, and the elevated plus maze. Anti-depressant effects of Plumeria obtusa L. were evaluated using the forced swim test (FST) and memory and learning were assessed using the Morris water maze (MWM) task. Po.Cr was also evaluated for phytochemicals using total phenolic content (TPC), total flavonoid content (TFC), and high-performance liquid chromatography assays, and antioxidant capability was assessed through assays of DPPH radical scavenging, total oxidation capacity, and total reducing capacity. In the alloxan-induced model of diabetes, the administration of Po.Cr and glibenclamide for 45days produced a marked decrease (p < 0.001) in hyperglycemia compared to control animals. Po.Cr treatment also resulted in improvement in indicators, such as body weight and lipid profile (p < 0.05), as well as restoration of normal levels of alanine transaminase (ALT) (p < 0.001), a biomarker of liver function. Diabetic rats presented more Alzheimer-like symptoms, with greater impairment of memory and learning, and increased anxiety and depression compared to non-diabetic normal rats, whereas treated diabetic rats showed significant improvements in memory and behavioral outcomes. These results demonstrate that Po.Cr reversed alloxan-induced hyperglycemia and ameliorated Alzheimer-related behavioral changes, which supports additional study and assessment of conventional use of the plant to treat diabetes and associated behavioral complications.

620. Pharmacokinetic-Pharmacodynamic Analysis of Oral Vancomycin and Gut Microbiome Changes in Healthy Volunteers: an Exploratory Study

Abstract Background Oral vancomycin causes profound changes to the gut microbiome due to high intra-colonic vancomycin concentrations. However, pharmacokinetics of oral vancomycin causing pharmacodynamic changes have not been explored, especially during the early dosing period. The purpose of this study was to investigate fecal vancomycin concentrations in healthy individuals in relation to gut microbiome diversity changes. Methods Healthy subjects 18-45 years with no antibiotic use for at least 28 days were given oral vancomycin 125 mg was given every 6 hours for 10 days. Stool samples were collected at baseline and during antibiotic therapy. Vancomycin concentrations were obtained through high-performance liquid chromatography (HPLC) assay. For this early pharmacokinetics analysis, stool samples from day 0 (baseline) to day 4 were included. Shotgun metagenomics sequencing was used for microbiome analysis. Descriptive analysis was performed to identify gut-microbiome phyla changes in correlation with detectable oral vancomycin fecal concentrations. Results A total of 6 healthy volunteers aged 32±5 years (Male: 100%; Caucasian: 50%; mean BMI: 26.8±4.5 kg/m2) were included. In the early dosing period (day 0-4), the mean fecal vancomycin concentrations increased daily with the highest concentration of 1,586 μg/g of stool occurring on day 4. Three of 9 subjects (50%) had undetectable oral vancomycin levels one days 1 and 2 of dosing. Within 24-48 hour of detectable vancomycin levels, subject-specific changes of gut microbiome phylum-level proportions were observable. Overall, an increase in Actinobacteria and Proteobacteria phyla and decrease in Firmicute phylum was observed within 24 hours that vancomycin was detected in the feces. Conclusion High concentrations of vancomycin are achieved in the stool by day four of dosing for all subjects; however, low concentrations are observed early in the dosing period for some subjects. Proportional, subject specific differences in gut microbiome phyla were observed within 24 hours of detectable vancomycin levels in the feces. Disclosures Kevin W. Garey, PharmD, MS, Acurx Pharmaceuticals: Grant/Research Support|Paratek Pharmaceuticals: Grant/Research Support|Seres Therapeutics: Grant/Research Support|Summit Pharmaceuticals: Grant/Research Support.

188: PHARMACOKINETIC ANALYSIS OF ANTIMICROBIAL THERAPY IN VENTRICULAR ASSIST DEVICE-SUPPORTED PATIENTS

Introduction: As a result of underlying heart failure, the utilization of left ventricular assist devices (VAD) has increased as a means of offloading the failing ventricle to improve cardiac output. Importantly, after device implantation and once end-organ perfusion has been enhanced, new pharmacokinetic alterations may acutely develop which may require pre-emptive dose adjustments for several pharmacotherapies. Currently, there are limited data in patients with VADs to determine patient and population-specific antimicrobial disposition. The purpose of this study is to assess perioperative pharmacokinetic changes in patients during VAD implantation receiving prophylactic antibiotics. Methods: Patients will be prospectively enrolled to collect blood samples peri-operatively and post-operatively. Blood samples will be used to analyze the antimicrobial concentrations using a validated high-performance liquid chromatography (HPLC-UV) assay. Results: To date, 11 patients have been enrolled in this study. Enrollees are on average 63 years of age, predominately male (82%), with nonischemic cardiomyopathy (64%). LVAD implantation consisted of HeartMate3 (n=9), and HVAD devices (n=2). Standard prophylactic regimens for LVAD implantation include ceftazidime and vancomycin. Intra-operatively, all patients received one prophylactic dose of ceftazidime 2 grams from which samples were collected thereafter. The median samples collected intra-operatively was 4 (IQR 3,6). Post-operatively, ceftazidime prophylaxis was continued for 36 hours, and samples were typically collected after the third ceftazidime dose. The median samples collected post-operatively was 4 (IQR 4,5). Antimicrobial plasma concentrations are currently pending processing and analysis. The drug concentration in the serum will be analyzed to determine the maximum concentration, area under the concentration-time profile for the dosing interval, half-life, clearance, and volume of distribution. Conclusions: In this medically complex heart failure patient population, use of narrow therapeutic index drugs can have detrimental consequences if they are not dosed appropriately. This study will provide practitioners more insight in perioperative dosing regimens for VAD patients by understanding how serum drug levels may be altered with VAD therapy.

The Effect of Storage at -20°C and -80°C on HbA1c Measurements in Whole Blood Samples Analysed Using Bio-Rad D-10

Introduction: Glycated haemoglobin (HbA1c) is a test commonly measured in many clinical laboratories to monitor and diagnose diabetes mellitus (DM). The laboratory in Hospital Tengku Ampuan Afzan (HTAA) receives HbA1c samples internally and from several other hospitals and clinics in the state of Pahang, at which, before transportation and analysis, the samples are often stored. This study determined the stability of HbA1c samples following different storage temperatures and duration for up to 30 days. Method: Whole blood samples for HbA1c analysis were collected from 222 healthy blood donors and type 2 DM (T2DM) patients. Each sample was prepared into four aliquots, which were then stored at temperatures -20°C and -80°C. HbA1c analyses were performed at baseline, days 15 and 30 using ion-exchange high-performance liquid chromatography (HPLC) assay technique on Bio-Rad D-10 analyser. HbA1c levels following sample storage were compared to the levels at baseline. Results: The baseline HbA1c (mean±SD) was 6.6±2.2%. At -20°C of storage, the HbA1c levels decreased with a mean difference of 0.1% and 0.3% on days 15 and 30, respectively. Storage at -80°C resulted in a mean difference of 0.1% on day 15 whilst no change was noted on day 30. The number of samples that showed clinically significant HbA1c change (defined as a change of &gt;0.5%) was less for the samples stored at -80°C (4.5%) as compared to -20°C (23%). Conclusion: HbA1c concentration is affected by temperature and duration of sample storage. In instances where prolonged storage of HbA1c samples is expected, storage at -80°C is preferable.

Evaluation of Bio-Rad D100 for HbA1c testing of dried blood spot samples

Abstract Hemoglobin A1c (HbA1c) testing is critical for diabetes diagnosis and monitoring. Although many studies have demonstrated the feasibility of measuring HbA1c from dried blood spots (DBS) using immunoassays, enzymatic assays, and certain high-performance liquid chromatography (HPLC) assays, there is limited evidence supporting the use of the Bio-Rad D100 HPLC method. We evaluated the feasibility of testing HbA1c from DBS using Bio-Rad D100 to support the assessment of glycemic control for the growing field of telehealth. Deidentified residual EDTA whole blood samples were processed, and HbA1c was measured on the Bio-Rad D100 on day zero. Concurrently, DBS were prepared for each sample by spotting 50 µL of blood onto a Whatman® 903 filter paper. The samples were dried overnight and stored at room temperature for two days. On day 2, a DBS disc with a diameter of 3.2 mm was eluted with 4.5 mL of Bio-Rad diluent for 20 minutes. The supernatants of the DBS samples were then processed on the platform. HbA1c values from the DBS samples were compared to the EDTA whole blood results. Compared to the EDTA samples, the HbA1c concentrations of the corresponding DBS samples were elevated, especially for samples with original results less than 7.0%. Furthermore, the chromatograms of the DBS samples were significantly altered; we observed baseline elevation and the appearance of unknown minor peaks. This issue was investigated. First, blank DBS samples were created by directly spotting filter paper with diluent or adding diluent into an empty EDTA tube and then spotting the filter paper with the mixture of diluent and EDTA. The blank DBS samples were tested, and the lack of peaks suggested that the interference was not from the diluent, EDTA, or the filter paper. DBS samples were also prepared with sodium heparin whole blood, finger-stick capillary blood, and multiple brands of filter papers and tested using a different HPLC method. However, the interference was still present in those samples, indicating the interference is most likely due to the time-dependent degradation of the samples themselves. Of note, the alternate HPLC method showed less chromatogram alteration. To prevent possible adduct formation, DBS samples were eluted in the presence of various concentrations of cysteine (Jeppsson et al. 1986). However, the pretreatment did not eliminate the chromatogram changes. Finally, a multiple regression model was created using the results of day two A1c, A0, and F peaks, and a correction factor was generated. The corrected HbA1c concentrations were well within +/-6% of the initial HbA1c concentrations. In conclusion, the Bio-Rad D100 HPLC method appears particularly susceptible to stability changes in DBS samples, and further analysis is needed before employing a correction factor.

KXS Balances the Tryptophan Metabolism in Mild to Moderate Depressed Patients and Chronic Restraint Stress Induced Depressive Rats.

Tryptophan metabolism is involved in the etiology and exacerbation of depressive disorders. Kai-Xin-San (KXS), a traditional Chinese medicine formula, has been widely used to treat depression and modulate serotonin simultaneously, but how it regulates depressive-like behavior by shifting the balance of the tryptophan-serotonin metabolism and kynurenine pathway remains vague. Ten participants with mild to moderate depression treated with KXS (KXS preparation) were analyzed in this study. Depression rating scale score and the concentration of serum tryptophan, 5-hydroxytryptophan and kynurenine was measured at baseline and the endpoint of KXS treatment. To explore the specific regulatory mechanism of KXS in tryptophan metabolism, the chronic restraint stress (CRS) was used to induce depressive-like syndrome in rats and the hippocampus level of tryptophan, 5-hydroxytryptophan, kynurenine with downstream metabolites (kynurenic acid, quinolinic acid) and key enzymes (indoleamine 2,3-dioxygenase, kynurenine 3-monooxygenase, kynurenine aminotransferase) were analyzed by liquid chromatography-electros pray ionization tandem mass spectrometry, high performance liquid chromatography and enzyme-linked immunosorbent assay respectively. KXS significantly decreased depression rating scale scores and increased the serum tryptophan and kynurenine concentration in depressive patients compared to baseline. Also, it alleviated the depressive behavior in CRS rats obviously. Comparing with CRS group, KXS increased tryptophan, 5-hydroxytryptophan, kynurenine level in rat hippocampus. Furthermore, in kynurenine pathway, KXS decreased the expression of indoleamine 2,3-dioxygenase, increased kynurenic acid by upregulating the expression of kynurenine aminotransferase while decreased quinolinic acid level in hippocampus, which suggested that KXS more favored improving serotonin pathway, and neuroprotective kynurenic acid branch in the tryptophan metabolism. This is the first tryptophan metabolomic study of patients with depression undergoing KXS treatment. Combining these clinical results with CRS induced rat model studies, it verified that KXS achieves an excellent antidepressant effect and balances tryptophan-kynurenine metabolic pathways by regulating some key metabolic products and enzymes.

Quantitation of Venetoclax in Human Plasma by High-Performance Liquid Chromatography with Ultraviolet Detection.

A simple, highly sensitive and specific method based on high-performance liquid chromatography (HPLC) with ultraviolet detection was developed for the measurement of venetoclax concentrations in plasma samples. The chromatographic method employed a mobile phase of acetonitrile: 0.5% KH2PO4 (pH3.5) (80/20, v/v) on a CAPCELL PAK C18 UG120 column at a flow rate of 0.5mL/min. The quantitative method was validated based on standards described in "Bioanalytical Method Validation: Guidance for Industry" published by the US Food and Drug Administration. The separation of venetoclax and the internal standard R051012 was satisfactory, and the chromatograms were free of interfering peaks from the biological matrix. The intra- and inter-day coefficients of variation for venetoclax assays were <12.9%, whereas intra- and inter-day accuracies were within 13.6%. Only 100μL of human plasma was required to detect a lower limit of quantification of 10ng/mL for venetoclax. The recoveries of venetoclax extracted with an Oasis HLB cartridge were between 81 and 85%. The developed HPLC method was successfully applied to the determination of venetoclax concentrations in plasma of acute myeloid leukemia patients taking venetoclax. The degree of drug interactions between venetoclax and CYP3A4 inhibitors can be determined by this HPLC assay.

Folate (vitamin B9) content analysis in bread wheat (Triticum aestivum L.)

Vitamin B9, particularly folic acid, is an essential molecule for human health. Wheat flour is one of the major sources of calorie intake by humans. The selection of folate-rich genotypes in wheat breeding can enhance the natural folate value in the daily diet. This study used a precise, high-performance liquid chromatography (HPLC) assay to analyze folate content in a 262-accession Chinese wheat mini-core collection (MCC) grown under three environments. Four folate derivatives in grains including tetrahydrofolate (THF), 5-methyltetrahydrofolate (5-CH3-THF), 5-formyltetrahydrofolate (5-CHO-THF), and 5,10-methenyltetrahydrofolate (5,10-CH+THF) were considered. An association analysis of water regimes, accession types, released years, geographical origin, and agronomic traits with folate content was conducted for the first time. There was a large amount of variation in folate content in the analyzed accessions, with genotype identified as the main influencing factor. Total folate content was significantly correlated with the content of the four MCC derivatives under the three environments. 5-CH3-THF and 5-CHO-THF were the most abundant among the four folate derivatives and were positively correlated with high folate content. The 12 accessions with the highest folate content showed an average of more than 80 μg/100 g. The analysis demonstrated that this Chinese wheat had not undergone extensive selection for folate content during breeding, which is unrelated to the geographical origin, accession types, winter/spring types, and grain colors of wheat. The content of THF, 5-CH3-THF, and 5,10-CH+THF was significantly negatively correlated with grain width, grain thickness, and thousand kernel weight. A relatively weak negative relationship manifested between folate contents and flowering date, whereas no significant correlation with tiller number, grain number per spike, maturity date, height, and spike length was detected. The investigation benefits wheat breeders for folate enhancement.

Comparison of odor and quality of Galli Gigerii Endothelium Corneum derived from domestic chickens and broilers

Galli Gigerii Endothelium Corneum(GGEC) is commonly used for the clinical treatment of indigestion, vomiting, diarrhea, and infantile malnutrition with accumulation. In recent decades, omnivorous domestic chickens, the original source of GGEC, has been replaced by broilers, which may lead to significant changes in the quality of the yielding GGEC. Through subjective and objective sensory evaluation, biological evaluation, and chemical analysis, this study compared the odor and quality between GGEC derived from domestic chickens and that from broilers. The odor intensity between them was compared by odor profile analysis and it was found that the fishy odor of GGEC derived from domestic chickens was significantly weaker than that of GGEC from broilers. Headspace-solid phase microextraction-gas chromatography-triple quadrupole tandem mass spectrometry(HS-SPME/GC-QQQ-MS/MS) suggested that the overall odor-causing chemicals were consistent with the fishy odor-causing chemicals. According to the odor activity va-lue and the orthogonal partial least squares discriminant analysis(OPLS-DA) result, dimethyl trisulfide, 2-methoxy-3-isobutylpyrazine, and 2-methylisoborneol were responsible for the fishy odor(OAV≥1) and the content of fishy odor-causing chemicals in GGEC derived from broilers was 1.12-2.13 folds that in GGEC from domestic chickens. The average pepsin potency in GGEC derived from broilers was 15.679 U·mg~(-1), and the corresponding figure for the medicinal from domestic chickens was 26.529 U·mg~(-1). The results of pre-column derivatization reverse-phase high-performance liquid chromatography(RP-HPLC) assay showed that the content of total amino acids and digestion-promoting amino acids in domestic chickens-derived GGEC was 1.12 times and 1.15 times that in GGEC from broilers, and the bitter amino acid content was 1.21 times folds that of the latter. In conclusion, GGEC derived from domestic chickens had weaker fishy odor, stronger enzyme activity, higher content of digestion-promoting amino acids, and stronger bitter taste than GGEC from broilers. This study lays a scientific basis for studying the quality variation of GGEC and provides a method for identifying high-quality GGEC. Therefore, it is of great significance for the development and cultivation of GGEC as both food and medicine and breeding of corresponding varieties.