Clinical Mass Spectrometry最新文献

Background

LC-MS/MS methods for multiplexed analysis of steroid hormones in saliva are useful research tools in endocrinology, but require sensitive detection.

Objective

To explore the use of hydrazide and hydrazine derivatization to improve sensitivity of detection for ketosteroids. On development and validation of a sample preparation method based on robot pipetting in the 96-well format we intended to then establish normal reference ranges for women and men, with saliva collected both in the early morning and late at night.

Method

Four hydrazides and one hydrazine were evaluated for their effectiveness as derivatization reagents based on the comparative signal response and efficacy of LC separation of five ketosteroid hydrazones via a methanol gradient mixed with either 0.2% formic acid or 0.1% ammonium hydroxide. Processing of saliva via liquid–liquid extraction (LLE) was optimized by examining variations in both extraction solvent polarity and protein precipitation reagents intended to inhibit emulsion.

Results

LLE using 10 % butanol in methyl tert-butyl ether (MTBE), with tannic acid as emulsion inhibitor, followed by 2-hydrazinopyridine (2-HP) derivatization enabled the multiplexed measurement of cortisol, cortisone, testosterone, dehydroepiandrosterone (DHEA), progesterone, and 17-alpha-hydroxyprogesterone (17-OHP) in the majority of saliva samples from women and men for this study.

Conclusion

Tannic acid is a novel and effective protein precipitation reagent in bioanalytical sample preparation. The validated method was used in the establishment of reference ranges, the success of which indicated that the method is suitable for Cushing’s screening and has potential as a novel analytical research tool.

A proteotyping approach using high resolution mass spectrometry has been applied, for the first time, to subtype the hepatitis C virus based upon detection of one or more signature peptides derived from the E1 and E2 envelope glycoproteins. These signature peptides represent conserved peptide segments within these proteins for particular subtypes of the virus that are found to be unique in mass when compared with the theoretical masses for all peptide segments of translated HCV proteins within a specifically constructed database. The successful application of the approach to three different subtypes of the virus (i.e., 1a, 1b and 2b) is demonstrated for protein and whole virus proteolytic digests. The approach has the potential to replace existing PCR-based subtyping by offering a more direct and cost comparable strategy that is not challenged by mixed infection scenarios.

Volumetric absorptive micro sampling (VAMS™) allows accurate sampling of 10 µL of blood from a minimally invasive finger prick and could enable remote personalized health monitoring. Moreover, VAMS overcomes effects from hematocrit and sample heterogeneity associated with dried blood spots (DBS). We describe the first application of VAMS with the Mitra® microsampling device for the quantification of protein biomarkers using an automated, high-throughput sample preparation method coupled with mass spectrometric (MS) detection.

The analytical performance of the developed workflow was evaluated for 10 peptides from six clinically relevant proteins: apolipoproteins A-I, B, C-I, C-III, E, and human serum albumin (HSA). Extraction recovery from blood with three different levels of hematocrit varied between 100% and 111% for all proteins. Within-day and total assay reproducibility (i.e., 5 replicates on 5 days) ranged between 3.2–10.4% and 3.4–12.6%, respectively. In addition, after 22 weeks of storage of the Mitra microsampling devices at −80 °C, all peptide responses were within ±15% deviation from the initial response. Application to data-independent acquisition (DIA) MS further demonstrated the potential for broad applicability and the general robustness of the automated workflow by reproducible detection of 1661 peptides from 423 proteins (average 15.7%CV (n = 3) in peptide abundance), correlating to peptide abundances in corresponding plasma (R = 0.8383).

In conclusion, we have developed an automated workflow for efficient extraction, digestion, and MS analysis of a variety of proteins in a fixed small volume of dried blood (i.e., 10 µL). This robust and high-throughput workflow will create manifold opportunities for the application of remote, personalized disease biomarker monitoring.

Background

The measurement of catecholamines and their metabolites in either urine or plasma is an important diagnostic test used to exclude the presence of neuroendocrine tumours. Because of weak chromatographic retention and potential ion-suppression, reverse-phase LC-MSMS is not ideal for analysis of these polar molecules. Here, we investigate derivatisation by ethylation as an alternative approach.

Methods

A simple and rapid method involving acetaldehyde and a reducing agent was used to convert urine free metanephrines and catecholamines, and their deuterated analogues as internal standards, to mono-ethyl or diethyl- derivatives. Using an Agilent 6460 triple-quadrupole mass spectrometer, precursor and product ion mass spectra were recorded to allow comparison of multiple reaction monitoring methods for both derivatised and non-derivatised analytes under reverse-phase LC-MSMS conditions with positive electrospray ionization.

Results

Conversion of biogenic amines to less polar ethyl derivatives increased their mass and enhanced the intensity of their molecular ions and fragments. Ethylation also improved the chromatographic properties of the amines, with greater retention and elution from reverse-phase HPLC columns with a methanol or acetonitrile gradient. The signal response of tandem mass spectrometric detection was increased up to 50-fold for ethyl metanephrines compared to non-derivatised compounds. This increase allowed for the omission of solid-phase extraction of urine as a clean-up step prior to analysis. The ‘dilute-derivatise-shoot’ method maintained analytical performance with respect to between-run imprecision (CV < 6%) and accuracy in an external quality assurance program. Gender-related ranges for free metanephrines in early-morning spot urines, collected from adult patients, were similar using either derivatised or non-derivatised samples.

Conclusions

The LC-MSMS detection of free urine biogenic amines can be greatly enhanced by ethyl derivatisation, which is easy and rapid to perform. Advantages include improved chromatography and lower limits of quantitation, that negate the requirement for solid-phase clean-up of urine prior to analysis. A disadvantage is the potential toxicity of the derivatising agents used if they are not handled appropriately.

Urine screening can be used to detect misuse of illicit drugs and validate opioid replacement therapy compliance. It is common that immunochemical assays are combined with GC-MS for these applications. Bruker has recently released an ion trap mass spectrometer, called Toxtyper™, with the potential to replace current screening algorithms to detect drug misuse.

Here, we compare our current strategy of urine screening for misuse of cannabis, amphetamines, cocaine, opiates, benzodiazepine, methadone, sufentanil, and pregabalin to the Toxtyper protocols provided by the manufacturer.

The analytical performance of the instrument was determined on a selected drug panel and with 188 urine samples being compared to establish concordance between our currently established approach and the Toxtyper.

The lower limits of detection and identification for acetylcodeine, amphetamine, benzoylecgonine, methadone, and nordiazepam were below the common cut-offs for immunological screening assays and comparable to GC-MS. Imprecision and accuracy, both within- and between-series, were consistently <25%. Toxtyper screening for pregabalin and sufentail was less sensitive than a targeted LC-MS/MS assay. Concordance met the predefined criterion of >90% for all drugs, except for pregabalin. Cannabis misuse could not be detected due to the limited sensitivity of the Toxtyper assay protocols used and the inherent imprecision of the assay.

Our study has revealed that a considerable portion of our current time-consuming protocol for screening drugs of abuse in urine, based on the combination of multiple analytical methods, could be consolidated by the Toxtyper for a majority of the most-relevant substances in our patient population.

Bile acids malabsorption (BAM) is encountered in numerous gastrointestinal pathologies and is a good example of a treatable cause of watery diarrhea after ileal resection. The gold standard for diagnosing BAM is the selenium homocholic acid taurine test (SeHCAT), an expensive and complex analysis. An alternative method is the quantification of 7α-hydroxy-4-cholesten-3-one (C4). Here, we present a simple, ultra high-performance liquid chromatography–tandem mass spectrometry method to measure C4 in human serum. To avoid time consuming sample preparation (e.g., derivatization, solid phase extraction), we used absorption chemistry-based extraction plates. This method demonstrates a lower limit of quantification of 5 ng/mL and is linear over a concentration range from 5 to 300 ng/mL (R² = 0.9977). Inaccuracy and imprecision were less than 15%. The validated method is currently used for routine measurement of C4 from serum in patients to confirm BAM diagnosis.

Mycophenolic acid (MPA) is the active metabolite of the immunosuppressant drug mycophenolate mofetil (MMF), which is commonly prescribed after organ transplantation in conjunction with other immunosuppressants. MMF therapy is monitored to balance therapeutic efficacy with minimizing adverse effects associated with high serum concentrations. A LC–MS/MS method was developed for the quantification of MPA and two additional metabolites, 7-O-mycophenolic acid glucuronide (MPAG) and mycophenolic acid acyl-glucuronide (AcMPAG), in serum using reverse-phase chromatography and multiple reaction monitoring (MRM) in positive electrospray ionization mode. Analytes were chromatographically resolved and the method was linear from 0.5 to 30.0 µg/ml MPA, 4.7 to 300 µg/ml MPAG, and from 0.5 to 30.0 µg/ml AcMPAG. Calibration curves for all analytes had r ≥ 0.990. Intra- and inter-assay imprecision coefficients of variation (CVs) were ≤6.9% and ≤14.5%, respectively. No ion suppression or interferences were observed. The method compared favorably with an unaffiliated reference laboratory. Retrospective data analyses indicate interpatient differences in drug metabolism.

A common method for identifying an unknown compound involves acquiring its mass spectrum and then comparing that spectrum against a spectral database, or library. Accurate comparison and identification is dependent on the quality of both the library and the test spectrum, but also the search algorithm used. Here, we describe a redesigned probability-based library search algorithm (ProLS) and compare its performance against two predicate algorithms, AMDIS from NIST (NIST) and LibraryView/MasterView (LV/MV), on human urine samples containing drugs of interest that were analyzed by quadrupole-time of flight (QqTOF) mass spectrometry. Each algorithm was used to compare the spectral data collected against an in-house spectral library. ProLS outperformed both NIST and LV/MV in efficiency of drug detection. Additionally, it demonstrated a scoring profile that resulted in an increased likelihood of low match scores for compounds that were absent from a sample. Increased scoring accuracy has the potential to reduce the time that analysts spend manually reviewing match data. Although search algorithms tend to be underappreciated, since they are not typically part of the end-user interface, this work illustrates how a redesigned algorithm can impact the accuracy of identification of small molecules in a biological matrix, and influence the overall utility of a bioanalytical method.