High-pressure Liquid Chromatographic Assay Research Articles

Detection of some bacteria and mycotoxins in the baladi chicken eggs from backyard in Bahira Governorate

Background Public health risks result from the consumption of baladi eggs due to the Egyptian consumer’s demand and preference for baladi chicken eggs that might be contaminated with bacteria and/or mycotoxins that might affect eggs quality and results in food-borne intoxication or infection to consumers. Objective The current study aimed to investigate the presence of certain bacteria (mainly Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia and Salmonella) and shed light on the virulence genes and also detection of mycotoxins in baladi agg content, which act as potential public health problems for providing safe eggs suitable for human consumption. Materials and methods A total of 150 Baladi chicken eggs from backyard were arbitrarily collected from El-Behera Governorate, Egypt at 2022–2023 for recognition of S. aureus, E. coli, K. pneumonia, Salmonella and mycotoxins. Molecular detection of some bacterial virulence genes by Polymerase Chain Reaction (PCR), including some enterotoxins and hemolysin genes for some S. aureus isolates as (sea, seb, sec, sed and see, hla and hlb) and E. coli isolates virulence genes for (shiga toxin1, shiga toxin2, eaeA and astA). Quantification of ochratoxin A (OTA) and aflatoxins (AFS) in the eggs by a low-cost, high-recovery fluorescence detector (FLD) in conjunction with a straightforward, specific, and created High-Pressure Liquid Chromatography (HPLC) assay in accordance with green chemistry. Results and conclusion Bacteriological examination revealed isolation of S. aureus, E. coli and K. pneumonia at percent 26.6%, 58%, and34%, respectively. It was found that aflatoxin B1 (AFB1) is the predominant detected toxins (18%), and OTA amount was found to be lower than the of aflatoxins found. It was found that AFs and OTA were determined to be below the maximum allowable threshold in 98% of the egg samples examined.

CSF dopamine is elevated in first-episode psychosis and associates to symptom severity and cognitive performance.

The hypothesis of dopamine dysfunction in psychosis has evolved since the mid-twentieth century. However, clinical support from biochemical analysis of the transmitter in patients is still missing. The present study assessed dopamine and related metabolites in the cerebrospinal fluid (CSF) of first-episode psychosis (FEP) subjects. Forty first-episode psychosis subjects and twenty healthy age-matched volunteers were recruited via the Karolinska Schizophrenia Project, a multidisciplinary research consortium that investigates the pathophysiology of schizophrenia. Psychopathology, disease severity, and cognitive performance were rated as well as cerebrospinal fluid concentrations of dopamine and related metabolites were measured using a sensitive high-pressure liquid chromatography assay. CSF dopamine was reliably detected in 50% of healthy controls and in 65% of first-episode psychosis subjects and significantly higher in first-episode psychosis subjects compared to age-matched healthy controls. No difference in CSF dopamine levels was observed between drug-naive subjects and subjects with short exposure to antipsychotics. The dopamine concentrations were positively associated with illness severity and deficits in executive functioning. Dopamine dysfunction has long been considered a cornerstone of the pathophysiology of schizophrenia, although biochemical support for elevated brain dopamine levels has been lacking. The results of the present study, showing that FEP subjects have increased CSF dopamine levels that correlate to disease symptoms, should fill the knowledge gap in this regard.

Serum Androgen Metabolites Correlate with Clinical Variables in African and European American Men with Localized, Therapy Naïve Prostate Cancer.

Dihydrotestosterone (DHT) and testosterone (T), which mediate androgen receptor nuclear translocation and target gene transcription, are crucial androgens and essential molecular triggers required for the proliferation and survival of prostate cancer cells. Therefore, androgen metabolism is commonly targeted in the treatment of prostate cancer. Using a high-pressure liquid chromatographic assay with tandem mass spectral detection, we determined the serum levels of metabolites produced during DHT/T biosynthesis in African American (AA) and European American (EA) men with localized, therapy naïve prostate cancer. Serum progesterone and related metabolites were significantly lower in AA men than in EA men, and these differences were associated with rapid disease progression. Multivariate analysis revealed significant differences between a subset of intermediate androgen metabolites between AA and EA men and between men with <=3 + 4 and >=4 + 3 Gleason score disease. AA men have a significantly higher frequency of single nucleotide polymorphisms in CYP11B1 and CYP11B2, enzymes that regulate corticosterone-aldosterone conversion. Finally, higher levels of T and pregnenolone were associated with a lower risk of progression-free survival only in AA men. This work provides new insight into androgen metabolism and racial disparities in prostate cancer by presenting evidence of dysregulated androgen biosynthesis in therapy naïve disease that correlates with clinical variables.

Development and validation of a new HPLC method for the analysis of a novel oral suspension formulation of 50 mg/ml ursodeoxycholic acid for newborns

AbstractObjectivesDrugs are developed for adults, making it difficult to find suitable treatments for children. Hospital pharmacy has developed alternatives to respond to this medical need. The objective of this study is to present a new liquid formulation of ursodeoxycholic acid (UDCA) at a concentration suitable for treatment of neonatal jaundice, and to introduce a novel high pressure liquid chromatography (HPLC) assay method.MethodsFour formulations have been developed using suspension vehicles due to the low solubility of the active ingredient, and different concentrations of excipient, xanthan gum, needed to facilitate resuspension. An HPLC method coupled to a diode array detector (DAD) has been developed. This method was used to analyze chemical and microbiologic stabilities, as well as physicochemical properties and palatability.ResultsAfter formulation was chosen, our new HPLC method assay was developed and validated for the quantification of chemical and microbiological stabilities of our product. Both parameters were stable over three months. Palatability has been improved thanks to the addition of universal suspension adjuvants. Odor, appearance and taste were judged pleasant despite a bitter aftertaste, with a persistence of the UDCA resuspension after one month.ConclusionsThree months after informing neonatal department about the availability of the drug, patients and caregivers are satisfied, and production campaigns are routinely planned.

Assay validation and determination of in vitro binding of mefloquine to plasma proteins from clinically normal and FIP-affected cats.

The antimalarial agent mefloquine is currently being investigated for its potential to inhibit feline coronavirus and feline calicivirus infections. A simple, high pressure liquid chromatography assay was developed to detect mefloquine plasma concentrations in feline plasma. The assay's lower limit of quantification was 250 ng/mL. The mean ± standard deviation intra- and inter-day precision expressed as coefficients of variation were 6.83 ± 1.75 and 5.33 ± 1.37%, respectively, whereas intra- and inter-day accuracy expressed as a percentage of the bias were 11.40 ± 3.73 and 10.59 ± 3.88%, respectively. Accordingly, this validated assay should prove valuable for future in vivo clinical trials of mefloquine as an antiviral agent against feline coronavirus and feline calicivirus. However, the proportion of mefloquine binding to feline plasma proteins has not been reported. The proportion of drug bound to plasma protein binding is an important concept when developing drug dosing regimens. As cats with feline infectious peritonitis (FIP) demonstrate altered concentrations of plasma proteins, the proportion of mefloquine binding to plasma proteins in both clinically normal cats and FIP-affected cats was also investigated. An in vitro method using rapid equilibrium dialysis demonstrated that mefloquine was highly plasma protein bound in both populations (on average > 99%).

Validation of HPLC and Enzyme-Linked Immunosorbent Assay (ELISA) Techniques for Detection and Quantification of Aflatoxins in Different Food Samples.

Background: In Jordan as in other worldwide countries, mycotoxins are considered a serious national problem in food supplies. As a result, almost all nations are setting and adopting different regulations targeting the control of mycotoxins levels in the domestic food supply, including the problem of reliable sampling and analysis methods. Objective: It is necessary to improve and give evidence of analytical abilities of laboratories within Jordan and developing countries enabling them to monitor mycotoxins effectively in food to overcome non-tariff obstacles. Methods: We analyzed 40 samples from wheat, corn, dried fig and dried coffee beans for total aflatoxin content using High Pressure Liquid Chromatography (HPLC) and Enzyme Linked Immunesorbent Assay (ELISA) methods. Results: 40% of samples from wheat, 60% from corn, 30% from dried fig, and 50% from dried coffee beans were found positive when speaking of total aflatoxins, with average values between 1.14 and 4.12 μg/kg. Obtained results allow considering all tested food samples as fit for human consumption if compared with the labeled regulatory limit of allowed aflatoxins in the European Union. In detail, the limit of detection and the limit of quantification for methods used in this study were significantly lower than the maximum limits established by the European Union. Highlights: The procedure used in this study is suitable for detection of mycotoxins at very low concentration.

Development and characterization of sandwich-type enzyme-linked aptamer assay for the detection of rongalite in food

Rongalite is an essentially strong carcinogen, which due to its properties as a bleaching and brightening, is illegally added to the food processing. In this study, a sandwich-type enzyme-linked aptamer assay (ELAA) is developed by using a rongalite-specific aptamer G02 modified fluorescein amidite (FAM) as a capture probe and aptamer C01 modified biotin as a signal element. In the presence of rongalite, the aptamer G02-rongalite-aptamer C01 complex is produced, and the absorbance value can be subsequently measured. The sandwich-type ELAA was shown to detect rongalite with high specificity and affinity, with a KD value of 19.91 ± 1.321 nM. In addition, the standard curve was established, with the limit of quantification (LOQ) for rongalite at 10 ng mL−1. By calculating the slope of the standard curve and the standard deviation of the blank values, the method detection limit (MDL) was 0.572 ng mL−1. Additionally, the accuracy of the sandwich-type ELAA was demonstrated in real food samples. Compared with high-pressure liquid chromatography (HPLC) assay, the sandwich-type ELAA can detect rongalite directly, and it has great advantages in pre-treatment, operation technique and cost. In short, our data suggest that the sandwich-type ELAA may be applicable as a molecular detection technique for rongalite.

Stability of azasetron-dexamethasone mixture for chemotherapy-induced nausea and vomiting administration.

Combination antiemetic therapy has become common practice for the prevention of nausea and vomiting caused by anticancer drugs. In this study, we investigated the stability of azasetron hydrochloride 0.1 mg/mL plus dexamethasone sodium phosphate 0.05, 0.1, or 0.2 mg/mL in 0.9% sodium chloride injection and stored in polyolefin bags and glass bottles over a period of 14 days at 4°C and 48 hours at 25°C. The stability studies were evaluated by visual inspection, pH measurement, and a high-pressure liquid chromatography assay of drug concentrations. During the study period, the concentration of each drug in the various solutions remained above 97% of the initial concentration at both 4°C and 25°C when protected from room light. Under the condition of 25°C with exposure to room light, the concentrations of both drugs were significantly lowered over 48 hours. The pH value decreased, and the color changed from colorless to pink. Our study demonstrates that the azasetron-dexamethasone mixture at a clinically relevant concentration seems to be stable for 48 hours at 25°C and for 14 days at 4°C when packaged in polyolefin bags or glass bottles and protected from room light. The room light is the main influential factor on stability. Clinicians should be aware that combinations of azasetron hydrochloride and dexamethasone sodium phosphate in solution with light exposure should be avoided.

Phytochemical Screening and Content Determination of Different Species of Genus Caesalpinia belonging to Different Origin with Antidiabetic Activity

Aim: The main aim of the study is to investigate the phytochemical screening of C. decapetala along with the content determination of different species of genus Caesalpinia with respect to their antidiabetic activity and identification of most bioactive species belonging to different origins. Methods: To achieve our goal different species of genus Caesalpinia collected from China and Pakistan were subjected to open column chromatography, High Pressure Liquid Chromatography (HPLC), antioxidant, and antidiabetic assays for evaluation. Results: From η-BuOH fraction of C. decapetala extract, eight compounds were isolated using open column chromatography and identified as apigenin-7-rhamnoside (1), 4-O-methylepisappanol (2), caesalpinol (3), daucosterol (4), astragalin (5), kaempferol (6), quercitrin (7), and naringin (8) using Nuclear Magnetic Resonance (NMR) spectroscopy. HPLC analysis of different species of genus Caesalpinia showed that the most active antidiabetic compound ‘quercitrin’ was present more in C. pulcherrima followed by decreasing order in C. sappan, C.decapetala, and C. bonduc. Conclusion: The results indicated that quercitrin is the most bioactive content and C. pulcherrima is most bioactive specie of China origin from genus Caesalpinia.

The influence of soluble polysaccharides derived from rice straw upon cellulase production by Trichoderma reesei

Abstract Acid-treated, alkali-treated and ball-milled rice straw was used as a substrate by Trichoderma reesei for cellulase production. Of the three pretreated straws, ball-milled straw provided the highest yield of cellulase, which was 1.3-fold higher than commercial cultures containing cellose. The FT-IR of the precipitate from rice straw with 70% ethanol showed an absorption peak typical of polysaccharides, while high pressure liquid chromatography assays of monosaccharide composition showed that glucose was the major component, while galactose, xylose, arabinose and fructose were present in much smaller quantities. The addition of soluble polysaccharide (SPS) derived from rice straw increased FPase activity by 1.5-fold and β-glucosidase activity by 2.3-fold, respectively, indicating the inductive effect of SPS upon cellulase production, especially upon the yield of β-glucosidase. Ball-milling influenced the molecular weight and monosaccharide composition of SPS and thus affected the performance of SPS in cellulase production. Finally, the crude enzymes produced in culture with SPS were used for saccharification and provided a 1.3-fold higher yield of glucose than that produced in culture without SPS. Our results indicate that SPS could represent a key factor in solving problems caused by the lack of β-glucosidase activity in enzymes produced by Trichoderma reesei .

Validation of mid-infrared spectroscopy for macronutrient analysis of human milk.

Human milk has considerable variation in its composition. Hence, the nutrient profile is only an estimate and can result in under- or over-estimation of the intake of preterm infants. Mid-infrared (MIR) spectroscopy is an evolving technique for analyzing human milk but needs validation before use in clinical practice. Human milk samples from 35 mothers delivering at 35 weeks to term gestation were analyzed for macronutrients by MIR spectroscopy and by standard laboratory methods using Kjeldahl assay for protein, Mojonnier assay for fat and high-pressure liquid chromatography assay for lactose. MIR analysis of the macronutrients in human milk correlated well with standard laboratory tests with intraclass correlation coefficients of 0.997 for fat, 0.839 for protein and 0.776 for lactose. Agreement between the two methods was excellent for fat, and moderate for protein and lactose (P<0.001). This methodological paper provides evidence that MIR spectroscopy can be used to analyze macronutrient composition of human milk. Agreement between the methodologies varies by macronutrient.

Compatibility of irinotecan-loaded DC Bead with different volumes and types of non-ionic contrast media

ObjectivesIrinotecan-loaded microspheres are used for simultaneous embolisation and chemotherapy of liver metastases of colorectal carcinoma. The aim of the study was to evaluate the compatibility of recently introduced DC BeadM1...

Stability of butorphanol-tropisetron mixtures in 0.9% sodium chloride injection for patient-controlled analgesia use.

Tropisetron is an adjuvant for butorphanol used in intravenous patient-controlled analgesia (PCA) and has been reported to provide superior pain control. It is efficacious in reducing the incidence of postoperative nausea and vomiting. However, this admixture is not available commercially and stability data applicable to hospital practice are limited. This study aimed to describe the drug compounding and evaluates the long-term (up to 14 days) stability of butorphanol and tropisetron in 0.9% sodium chloride injection for PCA use.In this study, commercial solutions of butorphanol tartrate and tropisetron hydrochloride were combined and further diluted with 0.9% sodium chloride injection to final concentrations of butorphanol tartrate 0.08 mg/mL and tropisetron hydrochloride 0.05 mg/mL. The polyolefin bags and glass bottles were stored at 4°C and 25°C for up to 14 days. The drug stabilities were determined by visual inspection, pH measurement, and high-pressure liquid chromatography assay of drug concentrations.The data obtained for admixtures prepared and stored at temperatures of 25°C and 4°C show the drugs have maintained at least 98% of the initial concentration. All solutions remained clear and colorless over the 14-day period, and the pH value did not change significantly.The results indicate that admixtures of butorphanol tartrate 0.08 mg/mL and tropisetron hydrochloride 0.05 mg/mL in 0.9% sodium chloride injection solution were stable for 14 days when stored in polyolefin bags or glass bottles at 4°C and 25°C and protected from light. The infusion is feasible for manufacturing in pharmacy aseptic units and can be stored for up to 14 days for routine use in PCA infusions.

Inositol Analysis by HPLC and Its Stability in Scavenged Sample Conditions.

Inositol is a 6-carbon sugar alcohol that has been shown in limited studies to reduce retinopathy of prematurity and chronic lung disease in premature newborns. Developmentally it has a high concentration in the fetus that decreases with gestational age. It is transported from the fetus to the mother across the placenta. Although studies are underway to determine inositol kinetics in premature newborns treated therapeutically, the effects of gestational age, age after birth, and feeding on inositol concentrations after birth have not been studied adequately in premature newborns. Such studies would minimize blood removal and trauma in preterm newborns by using plasma samples scavenged from the clinical laboratory to measure inositol after birth, if they remain stable. This report describes a new high pressure liquid chromatographic assay for inositol and its use to study the stability of inositol in conditions of storage that might be encountered within the clinical laboratory. The assay is linear from 0 to 1000 Mm with a lower limit of quantitation of 50 μM. Inositol in human plasma remains stable in refrigeration and at room temperature for up to 14 days and is not affected by storage in red blood cells that are intact or lysed. Anticoagulants encountered in clinical blood samples do not interfere with the chromatograms. Thus, it is feasible to measure the changes in inositol concentrations in plasma from preterm newborns that is scavenged from the clinical laboratory after storage for as long as 14 days.

Butorphanol and ketamine combined in infusion solutions for patient-controlled analgesia administration: a long-term stability study.

BackgroundKetamine in subanesthetic dose added to butorphanol has been reported to give superior pain control when used for intravenous patient-controlled analgesia (PCA) after surgery. However, this admixture is not available commercially and stability data applicable to hospital practice are limited.Material/MethodsThe butorphanol-ketamine admixtures were prepared in polyolefin bags and stored in the dark at 4°C, 25°C, or 37°C for 15 days. The initial concentrations were 50–150 microgram/ml for butorphanol and 1–4 mg/ml for ketamine, respectively. The stabilities were determined by visual inspection, pH measurement, and high-pressure liquid chromatography (HPLC) assay of drug concentrations.ResultsOver the 15 days, all solutions were clear in appearance, and no color change or precipitation was observed among the three temperatures. The percentages of initial concentration of each drug were over 95% during the study period, and the pH value did not change significantly.ConclusionsThe results indicate that the drug mixtures of butorphanol and ketamine in 0.9% sodium chloride injection were stable for 15 days when stored in polyolefin bags at 4°C, 25°C, or 37°C.

Liquid chromatography tandem mass spectrometry in the clinical laboratory.

Clinical laboratory medicine has seen the introduction and evolution of liquid chromatography tandem mass spectrometry in routine clinical laboratories over the last 10-15 years. There still exists a wide diversity of assays from very esoteric and highly specialist manual assays to more simplified kit-based assays. The technology is not static as manufacturers are continually making improvements. Mass spectrometry is now commonly used in several areas of diagnostics including therapeutic drug monitoring, toxicology, endocrinology, paediatrics and microbiology. Some of the most high throughput analyses or common analytes include vitamin D, immunosuppressant monitoring, androgen measurement and newborn screening. It also offers flexibility for the measurement of analytes in a variety of different matrices which would prove difficult with immunoassays. Unlike immunoassays or high-pressure liquid chromatography assays using ultraviolet or fluorescence detection, mass spectrometry offers better specificity and reduced interferences if attention is paid to potential isobaric compounds. Furthermore, multiplexing, which enables multiple analytes to be measured with the same volume of serum is advantageous, and the requirement for large sample volumes is decreasing as instrument sensitivity increases. There are many emerging applications in the literature. Using mass spectrometry to identify novel isoforms or modified peptides is possible as is quantification of proteins and peptides, with or without protein digests. Future developments by the manufacturers may also include mechanisms to improve the throughput of samples and strategies to decrease the level of skill required by the operators.

Electrospun vancomycin-loaded coating on titanium implants for the prevention of implant-associated infections.

The objectives of this work were to develop an antibiotic coating on the surface of a titanium plate to determine its antibacterial properties in vitro and in vivo. To prepare vancomycin-coated titanium implants, we adopted the electrospinning nanotechnique. The surface structure of the coating implants was observed using a scanning electron microscope. An elution method and a high-pressure liquid chromatography assay were used to characterize the release behavior of vancomycin from the coating. The antibacterial efficacy and the cytotoxicity of the coated titanium implants on osteoblasts were investigated in vitro. In addition, X-ray, white blood cell count, C-reactive protein, erythrocyte sedimentation rate, and pathological examination were performed to validate its antimicrobial efficacy in vivo. The antibiotic coating released 82.7% (approximately 528.2 μg) of total vancomycin loading in the coating in vitro. The release behavior of vancomycin from nanofiber coatings exhibited a biphasic release pattern with an initial burst on day 1, followed by a slow and controlled release over 28 days. There was no cytotoxicity observed in vitro for the vancomycin-loaded coating. The vancomycin-coated titanium implants were active in treating implant-associated infection in vivo. Thus, vancomycin-coated titanium implants may be a promising approach to prevent and treat implant-associated infections.

Stability of an epidural analgesic admixture containing butorphanol tartrate and ropivacaine hydrochloride

BackgroundLocal anaesthetics and opioid mixtures are commonly used for the management of postoperative pain. The purpose of this study was to investigate the stability of butorphanol tartrate and ropivacaine hydrochloride...

Impact of AhR, CYP1A1 and GSTM1 Genetic Polymorphisms on TP53 R273G Mutations in Individuals Exposed to Polycyclic Aromatic Hydrocarbons

This study was to undertaken to investigate the impacts of AhR, CYP1A1, GSTM1 genetic polymorphisms on the R273G mutation in exon 8 of the tumor suppressor p53 gene (TP53) among polycyclic aromatic hydrocarbons (PAHs) exposed to coke-oven workers. One hundred thirteen workers exposed to PAH and 82 control workers were recruited. We genotyped for polymorphisms in the AhR, CYP1A1, GSTM1, and TP53 R273G mutation in blood by PCR methods, and determined the levels of 1-hydroxypyrene as PAH exposure marker in urine using the high pressure liquid chromatography assay. We found that the distribution of alcohol users and the urinary excretion of 1-OHP in the exposed workers were significantly higher than that of the control workers (p=0.004, p<0.001, respectively). Significant differences were observed in the p53 genotype distributions of smoking subjects (p=0.01, 95%CI: 1.23-6.01) and PAH exposure (p=0.008, 95%CI: 1.24-4.48), respectively. Further, significant differences were observed in the p53 exon 8 mutations for the genetic polymorphisms of Lys/Arg for AhR (p=0.02, 95%CI: 0.70-15.86), Val/Val for CYP1A1 (p=0.04, 95%CI: 0.98-19.09) and null for GSTM1 (p=0.02, 95%CI: 1.19-6.26), respectively. Our findings indicated that polymorphisms of PAH metabolic genes, such as AhR, CYP1A1, GSTM1 polymorphisms may interact with p53 genetic variants and may contribute to PAH related cancers.

The mechanism of neurogenic pulmonary edema in epilepsy

Neurogenic pulmonary edema (NPE) is found in many epilepsy patients at autopsy. It is a life-threatening complication, known for almost 100years, but its etiopathogenesis is still not completely understood. In this study, we used the tremor rat (TRM: tm/tm) as an animal model of epilepsy to investigate the potential mechanisms of NPE under epileptic conditions. We performed reverse-phase high-pressure liquid chromatography assay, H&E and Masson staining, TUNEL assay, and Western blot experiments to determine the role of seizures in NPE. We found the level of catecholamine was higher in TRM rats. Also the occurrence of alveolar cell apoptosis was increased. Moreover, pulmonary vascular remodeling including the deposition of collagen and medial thickening was also found in TRM rats. Further study showed that cell apoptosis was mediated by increasing Bax, decreasing Bcl-2, and activating caspase-3. In addition, the protein level of phosphorylated ERK (p-ERK) was found to be decreased while phosphorylated JNK and phosphorylated p38 were upregulated in TRM rats. Thus, these findings suggest that pulmonary vascular remodeling and alveolar cell apoptosis might be involved in epilepsy-induced NPE and that the mitogen-activated protein kinase signal pathway was involved.