scholarly journals LC-MS/MS for Identifying Patients with CYP24A1 Mutations

2016 ◽  
Vol 62 (1) ◽  
pp. 236-242 ◽  
Author(s):  
Hemamalini Ketha ◽  
Rajiv Kumar ◽  
Ravinder J Singh
Keyword(s):  
Genetic Testing ◽  
Solid Phase ◽  
Internal Standard ◽  
Measurement Range ◽  
High Ratio ◽  
Unknown Etiology ◽  
Loss Of Function ◽  
Serum Samples ◽  
Dihydroxyvitamin D ◽  
Cytochrome P450 Family

Abstract BACKGROUND Patients have been described with loss-of-function CYP24A1 (cytochrome P450, family 24, subfamily A, polypeptide 1) mutations that cause a high ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D [25(OH)D/24,25(OH)2D], increased serum 1,25-dihydroxyvitamin D, and resulting hypercalcemia, hypercalciuria and nephrolithiasis. A 25(OH)D/24,25(OH)2D ratio that can identify patients who are candidates for confirmatory CYP24A1 genetic testing would be valuable. We validated an LC-MS/MS assay for 24,25(OH)2D (D3 and D2) and determined a 25(OH)D/24,25(OH)2D cutoff to identify candidates for confirmatory genetic testing. METHODS After addition of isotope-labeled internal standard, serum samples were extracted by solid-phase extraction, derivatized with 4-phenyl-1,2,4,-triazoline-3,5-dione, and quantified by LC-MS/MS. We measured 25(OH)D/24,25(OH)2D in 91 healthy patients and 34 patients with clinically suspected CYP24A1-mediated hypercalcemia. RESULTS The limits of detection and quantification were 0.03 (0.2) and 0.1 (0.24) nmol/L, respectively, for 24,25(OH)2D3, and 0.1 (0.23) and 0.5 (1.16) nmol/L for 24,25(OH)2D2. Intra- and interassay imprecision was 4%–15% across the analytical measurement range of 0.1–25 ng/mL (0.2–60 nmol/L). No interference was observed with 25(OH)D and 1,25(OH)2D. 25(OH)D/24,25(OH)2D of 7–35 was observed in healthy patients, whereas in 2 patients with CYP24A1 mutations, 25(OH)D/24,25(OH)2D was significantly increased (99–467; P < 0.001). A 25(OH)D/24,25(OH)2D ratio ≥99 identified patients who were candidates for CYP24A1 genetic testing. CONCLUSIONS Increased 25(OH)D/24,25(OH)2D supports the diagnosis of reduced CYP24A1 activity due to mutations in CYP24A1. Measurement of 25(OH)D/24,25(OH)2D should be considered a part of the clinical workup in patients with hypercalcemia of otherwise unknown etiology.

scholarly journals Quantitative Determination of Azithromycin in Human Plasma by Liquid Chromatography–Mass Spectrometry and its Application in Pharmackokinetic Study

2012 ◽  
Vol 11 (1) ◽  
pp. 55-63 ◽  
Author(s):  
Maizbha Uddin Ahmed ◽  
Mohammad Safiqul Islam ◽  
Tasmin Ara Sultana ◽  
AGM Mostofa ◽  
Muhammad Shahdaat Bin Sayeed ◽  
...  
Keyword(s):  
Mobile Phase ◽  
Solid Phase ◽  
Internal Standard ◽  
Extraction Process ◽  
Serum Samples ◽  
Limit Of Quantification ◽  
Relative Standard ◽  
Linear Concentration Range ◽  
Linear Concentration

Azithromycin is an effective and well-known antimicrobial agent. In the present study, a simple, sensitive and specific LC/MS/MS method has been developed and validated for the quantification of Azithromycin in  human serum samples using Clarithromycin as internal standard. Azithromycin was extracted from biological matrix  by using solid phase extraction process. The chromatographic separation was performed on Luna C18 (3 ?, 2x150   mm) column with a mobile phase consisting of 35 mM ammonium acetate buffer (mobile phase-A) and acetonitrile  and methanol in ratio of 90:10 ( as mobile phase-B) at a flow rate of 0.25 mL/min. The method was validated over a  linear concentration range of 0.5?50.0 ng/mL and limit of quantification (LOQ) was 0.5 ng/mL with a coefficient of  correlation (r2) = 0.9998. The intra-day and inter-day precision expressed as relative standard deviation were 1.64% – 8.43% and 2.32% – 9.92%, respectively. The average recovery of azithromycin from serum was 98.11%. The method  was successfully applied to a pharmacokinetic study after oral administration of Azithromycin 200 mg/5 ml suspension in healthy Bangladeshi volunteers. DOI: http://dx.doi.org/10.3329/dujps.v11i1.12488 Dhaka Univ. J. Pharm. Sci. 11(1): 55-63, 2012 (June)

scholarly journals Evaluation of a 1,25-Dihydroxyvitamin D Enzyme Immunoassay

2007 ◽  
Vol 53 (6) ◽  
pp. 1104-1108 ◽  
Author(s):  
Isolde Seiden-Long ◽  
Reinhold Vieth
Keyword(s):  
Enzyme Immunoassay ◽  
External Quality Assessment ◽  
Solid Phase ◽  
Colorimetric Detection ◽  
Serum Samples ◽  
External Quality Assessment Scheme ◽  
Dihydroxyvitamin D ◽  
1,25 Dihydroxyvitamin D ◽  
Receptor Assay ◽  
Calf Thymus

Abstract Background: Radioactive reagents are used in most assays for measurement of 1,25-dihydroxyvitamin D [1,25(OH)2D]. We evaluated a 1,25(OH)2D enzyme immunoassay (EIA) from IDS Ltd. that uses solid-phase immunoextraction and colorimetric detection and compared results to those of the thymus radioreceptor assay (RRA) for 1,25(OH)2D. Methods: We collected serum samples (n = 145) representing an even distribution (0–200 pmol/L) of 1,25(OH)2D concentrations and Vitamin D External Quality Assessment Scheme (DEQAS) proficiency survey samples from 2004 surveys (n = 15) and stored them at −20 °C. We analyzed all samples with both EIA and RRA methods. We calculated imprecision using 5 QC samples in quadruplicate in each run (n = 6), including both pooled patient material used for QC with the RRA and QC material included in the EIA reagent set. We evaluated calibration stability by analyzing calibrators from different lots on the same plate and determining if calculated sample values drifted significantly. Results: Deming linear regression between IDS EIA and RRA methods yielded slope 1.25 (95% CI 1.13–1.37), y-intercept −3 (95% CI −18 to 12), R2 = 0.74. DEQAS proficiency survey samples for 2004 were all within 30% of the all-methods-trimmed mean. Imprecision CVs were 12%–16% within-run and 15%–20% between-run. Conclusions: We find no evidence of inferiority to the classic calf-thymus receptor assay for 1,25(OH)2D and no disadvantage in the results generated by the IDS EIA using samples from the major proficiency survey for 1,25(OH)2D. According to the product insert, however, the IDS EIA underestimates 1,25(OH)2D2 compared with the D3 form.

scholarly journals Determination of Trace Zearalenone and Its Metabolites in Human Serum by a High-Throughput UPLC-MS/MS Analysis

2019 ◽  
Vol 9 (4) ◽  
pp. 741 ◽  
Author(s):  
Danlei Sun ◽  
Chenglong Li ◽  
Shuang Zhou ◽  
Yunfeng Zhao ◽  
Yun Gong ◽  
...  
Keyword(s):  
Human Serum ◽  
High Throughput ◽  
Solid Phase ◽  
Internal Standard ◽  
Ultra Performance Liquid Chromatography ◽  
Enzyme Hydrolysis ◽  
Serum Samples ◽  
Relative Standard ◽  
Before And After

This paper described an improved method for high-throughput and sensitive determination of zearalenone and its five metabolites (zearalanone, α-zearalenol, β-zearalenol, α-zearalanol and β-zearalanol) in human serum. Serum samples were measured both before and after enzyme hydrolysis to assess the free and total amount of each compound by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) in multi reaction monitoring (MRM) mode following off-line 96-well μElution solid-phase extraction (SPE). All the analytes were completely separated on a C18 column within 6 min. It enabled multi-sample preparation at the same time eliminating tedious evaporation and reconstitution steps, allowing 96 (one plate) samples to be processed and analyzed within 24 h. Using an isotope labelled internal standard (13C-ZEN), high recoveries were achieved for all the compounds in the range 91.6%–119.5%, with intra-day and inter-day relative standard deviations (RSDs) of less than 8%. The limits of detection (LOD) and the limits of quantification (LOQ) were 0.02–0.06 ng mL−1 (0.6–2 fmol) and 0.1–0.2 ng mL−1 (3–6 fmol), respectively, demonstrating a notable enhancement in sensitivity compared to the existing methods. The validated method was applied to the analysis of paired urine and serum samples collected from 125 healthy individuals in Henan Province, locating in the middle area of China. ZEN metabolites in human serum were significantly lower than those in urine. Only one serum sample was positive for ZEN after enzyme digestion, whereas at least one of ZEN biomarkers was detected in 75.2% of the paired urine samples. Some comparison and discussion were also included in this paper.

Analysis for Fully Oxidized Neopterin in Serum by High-Performance Liquid Chromatography

1992 ◽  
Vol 38 (9) ◽  
pp. 1722-1724 ◽  
Author(s):  
J J Rippin
Keyword(s):  
High Performance ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Extraction Procedure ◽  
Internal Standard ◽  
Marrow Transplant ◽  
Ammonia Solution ◽  
Ferric Nitrate ◽  
Serum Samples ◽  
The Mean

Abstract Fully oxidized D-neopterin in serum can be measured by HPLC. Serum samples were preincubated with ferric nitrate/EDTA solution to remove any dihydroneopterin, which is unstable and may give spuriously high results because of its conversion to D-neopterin. Pterins were extracted onto solid-phase propylbenzenesulfonic acid minicolumns and eluted with a 1:5 (by vol) mixture of ammonia solution (308 g/L) in acetonitrile. Extracts were evaporated and then reconstituted in mobile phase (50 mL of methanol per liter of 50 mmol/L phosphate buffer, pH 6.2) before injection. Separation was performed with a 25-cm ODS2 column (particle size, 5 microns) at 32 degrees C with fluorescence detection (lambda ex 360 nm, lambda em 440 nm). The between-batch CV was 7.1% and 5.6% for neopterin concentrations of 21.7 and 67.3 nmol/L, respectively. The limit of detection was 0.75 nmol/L, and the mean recovery of the extraction procedure was 90% for neopterin and internal standard. Correlation with a radioimmunoassay (x) gave y = 0.99x + 0.64 (r = 0.970, Sy/x = 2.75). The method allows daily analysis of serum D-neopterin in small batches and is currently used to monitor patients undergoing bone-marrow transplant.

scholarly journals Comparison of Capillary Electrophoresis with HPLC for Diagnosis of Factitious Hypoglycemia

2000 ◽  
Vol 46 (11) ◽  
pp. 1773-1780 ◽  
Author(s):  
Rita Paroni ◽  
Barbara Comuzzi ◽  
Cinzia Arcelloni ◽  
Stefano Brocco ◽  
Saula de Kreutzenberg ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Solid Phase ◽  
Clinical Chemistry ◽  
Internal Standard ◽  
Sodium Cholate ◽  
Hypoglycemic Agents ◽  
Serum Samples ◽  
Reduced Pressure ◽  
Electrokinetic Capillary Chromatography ◽  
Sulfonylurea Drugs

Abstract Background: The diagnosis of “factitious hypoglycemia” is essentially based on the disclosure of hypoglycemic agents in blood or urine. The aim of this study was to evaluate the performance of capillary electrophoresis (CE) as a quantitative method for determination of chlorpropamide, tolbutamide, glipizide, gliclazide, and glibenclamide in serum. Methods: Serum samples (1 mL), with internal standard added, were purified by solid-phase extraction on OASISTM HLB cartridges (Waters), dried under reduced pressure, and reconstituted with 30–60 μL of acetonitrile:H2O. Analysis was carried out by micellar electrokinetic capillary chromatography in 5 mmol/L borate, 5 mmol/L phosphate, 75 mmol/L sodium cholate, pH 8.5, containing 25 mL/L methanol. Separation was accomplished in a 20 cm × 50 μm (i.d.) silica capillary at 25 °C and a constant voltage of +10 kV. Pharmacokinetics of gliclazide (80-mg tablet) in a diabetic patient were assayed by both HPLC and CE. Two hypoglycemic patients positive by HPLC analysis for unreported gliclazide and tolbutamide overdose were also screened by CE. Results: Separation of six drugs (including the internal standard) was accomplished in 5 min plus 5 min rinsing. The between-day CV of the ratio of the areas of the sulfonylurea drugs to internal standard was <1% (n = 10). Linearity (r2 ≥0.998) and recovery (≥80%) were good for all sulfonylurea drugs tested. Pharmacokinetic curves for gliclazide by CE and HPLC were superimposable. CE analysis confirmed the HPLC diagnosis of surreptitious abuse of gliclazide and tolbutamide. Conclusion: CE is a useful tool in the clinical chemistry and toxicology laboratory for drug monitoring and pharmacokinetic investigations.

scholarly journals Detection of Persistent Organic Pollutants in Blood Serum of Electricians and Welders in Latvia

2016 ◽  
Vol 70 (5) ◽  
pp. 330-335
Author(s):  
Pāvels Sudmalis ◽  
Mārīte Ārija Baķe ◽  
Juris Rotbergs
Keyword(s):  
Blood Serum ◽  
Organic Pollutants ◽  
Persistent Organic Pollutants ◽  
Solid Phase ◽  
Full Range ◽  
Internal Standard ◽  
Control Group ◽  
Serum Samples ◽  
Chlorinated Pesticides ◽  
Mean Values

Abstract Persistent organic pollutants (POPs) are chemical substances that persist in the environment, bioaccumulate through the food web, and pose a risk of causing adverse effects to human health and the environment. The aim of the study was to assess the POP (polychlorinated biphenyls (PCBs), polybrominated diphenylethers (PBDEs), DDT and their derivatives) levels in blood serum to identify possible risk group workers. Blood serum samples (116 in total) were collected from two groups of employees — electricians, who can come in contact with PCB-containing transformer and capacitor oil, and welders, who were used as a control group. Sample purification was done by double solid phase extraction. The concentrations of POPs in blood serum were determined by gas chromatography with electron capture detector GC/ECD and recovery controlled by internal standard CB-174. None of the 116 samples contained the full range of tested POPs. However, all samples contained at least one of pesticides, and a marker PCB and mono-ortho PCB. Blood serum samples of 52% of electricians and 97.8% of welders contained non-ortho PCB compared to 84% and 74.7%, respectively, for PBDE’s. The concentrations of 18 detected PCBs, 4 detected PBDEs and 6 chlorinated pesticides and their metabolites varied in wide ranges and the differences in mean values between groups were not statistically significant (p > 0.05). The estimated concentrations of POPs correspond to the lowest levels detected in other countries. Mean concentrations of low-chlorinated marker PCBs were higher in the electrician group, suggesting that the employment sites are contaminated with PCBs, or that employees have contact with PCB-containing items.

Liquid chromatographic determination of meloxicam in serum after solid phase extraction

2010 ◽  
Vol 64 (4) ◽  
Author(s):  
Radia Ouarezki ◽  
Moulay-Hassane Guermouche
Keyword(s):  
Solid Phase Extraction ◽  
Solid Phase ◽  
Internal Standard ◽  
Chromatographic Determination ◽  
Array Detector ◽  
Aqueous Acetic Acid ◽  
Phase Extraction ◽  
Serum Samples ◽  
Liquid Chromatographic

AbstractA liquid chromatographic method for the determination of meloxicam in serum has been developed. The technique includes a solid phase extraction of the serum samples on [poly (divinylbenzeneco-N-vinylpyrrolidone)] as a solid phase extraction sorbent. After conditioning, the cartridge was loaded with 1 mL of acidified serum containing an internal standard. Elution was carried out using 1 mL of water-acetonitrile (φ r = 1: 1) mixture. After evaporation of the eluate to dryness and reconstitution of the residue with 0.1 mL of methanol, the samples were analyzed on a Symmetry C18 column. Mobile phase consisted of 1 % aqueous acetic acid/THF/acetonitrile (φ r = 60: 30: 10) + 0.1 mL of 1-octane sulfonic acid. Detection was carried out using a photodiode array detector. Full validation of the proposed method is provided. Linearity of the method was proven over the range of 0.01–10 φg mL−1 of meloxicam. Meloxicam assay was accurate and reliable with average intra- and inter-day precisions lower than 5.0 % and the intra- and inter-day accuracy higher than 97 %. Limits of detection (LOD) and quantitation (LOQ) found were 0.003 μg mL−1 and 0.01 μg mL−1, respectively. The proposed method was successfully utilized to quantify meloxicam in serum.

Evaluation of Serum Calprotectin Level and Disease Activity in Patients with Rheumatoid Arthritis

2019 ◽  
Vol 15 (4) ◽  
pp. 316-320
Author(s):  
Mir Amir Aghdashi ◽  
Seyedmostafa Seyedmardani ◽  
Sholeh Ghasemi ◽  
Zohre Khodamoradi
Keyword(s):  
Rheumatoid Arthritis ◽  
Disease Activity ◽  
Inflammatory Arthritis ◽  
Unknown Etiology ◽  
Tender Joint Count ◽  
Serum Samples ◽  
Tender Joint ◽  
Cross Sectional ◽  
Calprotectin Level ◽  
Remission Phase

Background: Rheumatoid Arthritis (RA) is the most common type of chronic inflammatory arthritis with unknown etiology marked by a symmetric, peripheral polyarthritis. Calprotectin also can be used as a biomarker of disease activity in inflammatory arthritis and other autoimmune diseases. Objective: In this study, we evaluated the association between serum calprotectin level and severity of RA activity. Methods: A cross-sectional study was conducted on 44 RA patients with disease flare-up. Serum samples were obtained from all patients to measure calprotectin, ESR, CRP prior to starting the treatment and after treatment period in the remission phase. Based on Disease Activity Score 28 (DAS28), disease activity was calculated. Results: Of 44 RA patients, 9(20.5%) were male and 35(79.5%) were female. The mean age of our cases was 53±1.6 years. Seventeen (38.6%) patients had moderate DAS28 and 27(61.4%) had high DAS28. The average level of calprotectin in the flare-up phase was 347.12±203.60 ng/ml and 188.04±23.58 ng/ml in the remission phase. We did not find any significant association between calprotectin and tender joint count (TJC; P=0.22), swollen joint count (SJC; P=0.87), and general health (GH; P=0.59), whereas significant associations were found between the calprotectin level and ESR (p=0.001) and DAS28 (p=0.02). The average calprotectin level in moderate DAS28 (275.21±217.96 ng/ml) was significantly lower than that in high DAS28 (392.4±183.88 ng/ml) (p=0.05). Conclusion: We showed that the serum level of calprotectin can be a useful and reliable biomarker in RA activity and its severity. It also can predict treatment response.

Bioanalytical method development and validation of simultaneous analysis of telmisartan and hydrochlorthiazide in human plasma using LC-MS/MS

2016 ◽  
Vol 5 (03) ◽  
pp. 4862 ◽  
Author(s):  
Mathew George* ◽  
Lincy Joseph ◽  
Arpit Kumar Jain ◽  
Anju V.
Keyword(s):  
Human Plasma ◽  
Retention Time ◽  
High Performance ◽  
Method Development ◽  
Matrix Effects ◽  
Solid Phase ◽  
Internal Standard ◽  
Assay Method ◽  
Buffer System ◽  
Internal Standards

A simple, sensitive, rapid and economic high performance thin layer chromatographic method and a mass spectroscopic assay method has been developed for the quantification of telmisartan and hydrochlorthiazide combination in human plasma. The internal standards and analytes were extracted from human plasma by solid-phase extraction with HLB Oasis1cc (30mg) catridges. The scanning and optimization for the samples are done using methanol: water (50:50). The samples were chromatographed using reverse phase chromatography with C-18 column of different manufacturers like Ascentis C18 (150×4. 6, 5µ) using the buffer system Acetonitrile: Buffer (80:20%v/v) which consist of 2±0. 1Mm ammonium format at a flow rate of 0. 7ml/min at a column oven temperature 35±10c. The internal standard used was hydrochlorthiazide13c1, d2 and telmisartand3. The extraction techniques include conditioning, loading, washing and elution, drying followed by reconstitution of the dried samples. The volume injected was 10µl with the retention time of 3-4 min for telmisartan, 1-2 min for hydrochlorthiazide and for the internal standards the retention time was 3-4 min for telmisartand3 and 1-2 min for hydrochlorthiazide c13d2. The rinsing solution was Acetonitrile: HPLC grade water in the ratio (50:50). The above developed method was validated using various parameters like selectivity and sensitivity, accuracy and precision, matrix effects, % recovery and various stability studies. The method was proved to be sensitive, accurate, precise and reproducible. The preparation showed high recovery for the quantitative determination of telmisartan and hydrochlorthiazide in human plasma.

scholarly journals UHPLC-ESI-MS/MS Quantification of Relevant Substrates and Metabolites of the Kynurenine Pathway Present in Serum and Peritoneal Fluid from Gastric Cancer Patients—Method Development and Validation

2021 ◽  
Vol 22 (13) ◽  
pp. 6972
Author(s):  
Ilona Sadok ◽  
Katarzyna Jędruchniewicz ◽  
Karol Rawicz-Pruszyński ◽  
Magdalena Staniszewska
Keyword(s):  
Gastric Cancer ◽  
Cancer Patients ◽  
Peritoneal Fluid ◽  
Method Development ◽  
Solid Phase ◽  
Kynurenine Pathway ◽  
Serum Samples ◽  
Esi Ms ◽  
Gastric Cancer Patients ◽  
Development And Validation

Metabolites and enzymes involved in the kynurenine pathway (KP) are highly promising targets for cancer treatment, including gastrointestinal tract diseases. Thus, accurate quantification of these compounds in body fluids becomes increasingly important. The aim of this study was the development and validation of the UHPLC-ESI-MS/MS methods for targeted quantification of biologically important KP substrates (tryptophan and nicotinamide) and metabolites(kynurenines) in samples of serum and peritoneal fluid from gastric cancer patients. The serum samples were simply pretreated with trichloroacetic acid to precipitate proteins. The peritoneal fluid was purified by solid-phase extraction before analysis. Validation was carried out for both matrices independently. Analysis of the samples from gastric cancer patients showed different accumulations of tryptophan and its metabolites in different biofluids of the same patient. The protocols will be used for the evaluation of tryptophan and kynurenines in blood and peritoneal fluid to determine correlation with the clinicopathological status of gastric cancer or the disease’s prognosis.

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