Clinical Mass Spectrometry最新文献

Hundreds of new psychoactive substances (NPS) covering most drugs-of-abuse classes have been introduced to the recreational drug market in recent years. One class of NPS drugs that has become more common recently is “designer” benzodiazepines. Due to a close structural resemblance with prescription benzodiazepines, some of these substances may elicit a positive response (i.e. cross react) in immunoassay screening. Consequently, it is increasingly important to include NPS benzodiazepines during method confirmation to ensure accurate identification of closely-related compounds as well as detection of the benzodiazepines themselves.

Here, we present our efforts to develop a screening and confirmation method for detection of 28 NPS benzodiazepines in urine using reversed-phase liquid chromatographic separation in combination with high-resolution mass spectrometry (LC–HRMS). MS was performed in positive electrospray mode on a Thermo Fischer Scientific Q Exactive Orbitrap instrument using either full scan (for screening) or parallel reaction monitoring (for confirmation).

We found the lower quantification limit of the method to range from 5 to 50 ng/mL. Analytical precision and accuracy were ≤15% for both screening and confirmation for all except one analyte. The method was used to analyze patient urine samples from routine drug testing and samples from acute intoxication cases presenting in emergency wards. Altogether, 16 of the 28 benzodiazepines (i.e., clobazam, clonazolam, deschloroetizolam, diclazepam, estazolam, etizolam, flubromazepam, flubromazolam, flunitrazolam, 3-hydroxyflubromazepam, 3-hydroxyphenazepam, ketazolam, meclonazepam, metizolam, nifoxipam, and pyrazolam) were detected in the urine samples.

The results from patient sample analysis indicate a high prevalence of NPS benzodiazepine use, emphasizing the importance of including novel drugs of abuse in drug testing menus.

Melanoma accounts for more than 60% of deaths associated with skin cancer, making its early detection through dermatological screening essential for improved treatment outcomes. Early detection and successful treatment of melanoma can dramatically increase the 5-year survival rate from 14 to 98%. To support such efforts, comprehensive identification of metabolite patterns capable of describing cancer progression will help support the foundational knowledge necessary to build early detection platforms for intervention prior to metastasis. Using an UPLC-MS, as part of a cell-based, untargeted metabolomics approach, we profiled the metabolomes of WM-226-4 and WM-115 cells. Derived from the metastatic and the primary sites of the same individual, these two cell lines represent a paired melanoma cancer cell line. Progenesis and MetaboAnalyst, platforms dedicated to metabolomics data analysis, were used to establish a panel of differentially expressed metabolites across these two stages of melanoma. In addition, mummichog was used to identify the affected pathways. A total of 12 differentially expressed metabolites including amino acids, carnitine, acylcarnitine, and a limited set of lipids were identified. The significantly differing metabolites are components of a diverse set of metabolic pathways (e.g., glycerophospholipid metabolism, carnitine shuttle, tryptophan metabolism), that have biological implications for the survival and dissemination of metastatic melanoma cells.

Here, volumetric absorptive microsampling (VAMS), used for the measurement of cardiovascular drugs, is compared against conventional dried blood spot (DBS) card sampling to evaluate adherence to prescribed medication. Volumetric absorptive microsampling (VAMS) is an attractive alternative to plasma sampling for routine drug monitoring and potentially overcomes haematocrit issues associated with quantitative bioanalysis of conventional dried blood spots. A quantitative VAMS-based LC-HRAM MS assay for atenolol, lisinopril, simvastatin and valsartan was developed and validated. The assay demonstrated acceptable linearity, selectivity, accuracy, precision, recovery and insignificant matrix effects with no impact of haematocrit on assay accuracy. Volunteers provided both VAMS and DBS 903 card samples (the current standard) to allow comparison of the two methods and demonstrate the potential utility of VAMS. Analysis of VAMS samples correctly identified drugs in volunteers known to be adherent, and found no false positives from volunteers known to be taking no medication. There was a strong correlation between the two sampling systems confirming the utility of VAMS. Therapeutic drug monitoring (TDM) can assist clinicians in deciding how to proceed with treatment in the event of poor improvement in patient health. VAMS could offer a potentially more efficient method of sample collection, with fewer rejected samples than the DBS approach.

In this study, a one-step method for liquid-liquid extraction has been compared against a two-step procedure for testosterone assays in terms of accuracy, specificity, recovery, lipid removal and baseline noise, using QCs and unknown samples. The difference in accuracy was less than 5% for adult sera, while it was less than 10% for prepubescent sera. To compare specificity, the ion ratio transition of 289 → 97 to 289 → 109 was monitored for all QCs and unknown samples; no interference in the testosterone peak was observed for any tested sample prepared by either the one-step or two-step procedure. The baseline comparison of LC–MS/MS chromatograms of samples indicated that samples prepared by the one-step procedure were of the same quality as those prepared by the two-step procedure; however, recovery in unaltered serum using the one-step procedure was approximately 15% greater across the low to high concentration range. Furthermore, recovery using the one-step procedure was more consistent between stripped and unstripped serum. Lipids were removed efficiently in the two-step procedure as verified by monitoring the typical phospholipid MRM transition of m/z 496 → 184. For every sample processed by the one-step procedure, a one-minute online column wash with 95% methanol was able to remove 95% of the bound lipids, thereby providing a column life-time approximately equivalent to that for the two-step procedure. The presented data indicate that the one-step procedure could replace the two-step procedure while maintaining accuracy, saving time, increasing recovery, and minimizing the potential for errors with the fewer steps required.

Drug monitoring of benzodiazepines is often performed in toxicology and pain management settings. During the validation of a laboratory-developed multiplex UPLC-MS/MS assay for quantifying benzodiazepines in human urine, we observed notable instability (greater than 20% nominal) in 7-aminoclonazepam (7AC) during prolonged freezing conditions by performing method comparisons and a conventional analyte stability study. These findings highlight the importance of proper calibrator storage and also the essential need to perform method comparison and analyte stability studies. It is recommended that clinical laboratories testing for urine 7AC carefully validate its stability if subjected to freezing conditions.

Background

Aldosterone measurement is critical for diagnosis of primary aldosteronism and disorders of the renin-angiotensin system. We developed an LC-MS/MS method for plasma and urinary aldosterone and compared it to our RIA method. We present a reference interval study for a Belgian population.

Methods

68 plasma and 23 urine samples were assayed for as part of a method comparison. For the reference interval study, we enrolled 282 healthy Caucasian volunteers (114 Male: mean age 35 ± 11 y and 168 Female: mean age 42 ± 13 y). A subset of 139 healthy volunteers agreed to a 24-h urine collection. For the method validation, 5 plasma and 8 urine pools were run in triplicate and quadruplicate, respectively, on 3 different days.

Results

Between-run imprecision (CV) was 2.8–5.1% for plasma and 4.5–8.6% for urine, except at the low urine concentration of 2.99 nmol/L where a CV of 15.4% was observed. The limit of quantitation was 0.04 nmol/L for plasma and 6.65 nmol/L for urine. Recoveries, based on spiking experiments into natural matrix, did not differ significantly from 100%. Regression comparisons showed that, on average, RIA generated results were 59% and 11% higher than LC-MS/MS for plasma and urine, respectively. The MS reference interval we propose for plasma aldosterone is 0.07 nmol/L–0.73 nmol/L for women and 0.04 nmol/L–0.41 nmol/L for men. No gender difference was observed for urine aldosterone. The reference interval was determined to be <60.94 nmol/day.

Conclusions

The LC-MS/MS method was validated and reference intervals for plasma and urine were established. A significant bias between RIA and LC-MS/MS was noted.

Multiplexed adrenal steroid measurement provides critical diagnostic information for patients with congenital adrenal hyperplasia (CAH) as confirmation of newborn screening (NBS) or as initial diagnosis. This study reports the implementation of an adrenal steroid profiling method with a turnaround time (TAT) of less than 24 h using liquid chromatography and tandem-mass spectrometry (LC-MS/MS). A lab-developed multiplexed LC-MS/MS assay was used to quantify levels of 11-deoxycortisol, cortisol, 17-hydroxy-progesterone (17-OHP), androstenedione, and testosterone. Intra and interassay imprecision were found to be <10%. Comparison with a reference laboratory revealed <20% bias for all 5 analytes and Deming correlation coefficients >0.990. Linearity ranges were established from the lowest to upper limit calibrator concentrations with 100- to 800-fold maximum dilution. Run to run carryover was <0.1%, and acceptable matrix effect was observed (i.e., ion suppression enhancement <15%). Compared to serum samples, ethylenediaminetetraacetic acid (EDTA) and heparin plasma had large positive bias in the measurement of 11-deoxycortisol (62.2% and 60.2%, respectively) and androstenedione (43.8% and 33.2%, respectively), while cortisol, 17-OHP and testosterone showed less than 20% bias between sample types. Hemoglobin, bilirubin, or triglyceride interference decreased 11-deoxycortisol measurement in EDTA plasma (−19.3%, −25.6%, and −25.0%, respectively). Lipemia increased the measurement of testosterone by 28.9%. In summary, our multiplexed LC-MS/MS method provided highly sensitive and specific measurement of adrenal steroids. EDTA, heparin, hemolysis, icterus and/or lipemia may significantly impact assay results and should be avoided. This method provides an effective strategy for improving TAT in CAH testing and confirmation of NBS results.

During the reproducibility validation for a time-of-flight (TOF) high-resolution mass spectrometry (HRMS) method set up to detect 61 drugs of abuse commonly encountered in the toxicology laboratory, it was noticed that, a number of compounds were not identified correctly during the between run analysis; the most difficult compounds to identify were norpropoxyphene, morphine, norbuprenorphine, nortriptyline, EDDP and tramadol. In subsequent patient comparison studies, screening a panel of 338 analytes, the TOF-HRMS method correctly identified 211 analytes over two runs, but did not identify 127. A total of 11 false positive results were identified by manual review of the data to be the result of confirmation ion signal-to-noise ratio(s) < 3, although one false positive that was difficult to resolve (i.e., identification of maprotiline as amitriptyline) was due to similar fragment ions and retention times. The TOF-HRMS method showed reasonable agreement with LC–MS/MS results, but there were a number of discrepant results. Additionally, the TOF-HRMS did detect five compounds missed by the LC–MS/MS methods. This extensive validation effort highlights the difficulty of analysis for certain compounds that are likely to require additional follow up prior to reporting a positive result, especially at low and high concentrations, regardless of the type of instrumentation involved.