Plasma Matrix Effects Research Articles

Strategies for the Immobilization and Signal Amplification of a Double Nanobody Sandwich ELISA for Human Microsomal Epoxide Hydrolase.

The microsomal epoxide hydrolase (mEH) is important in the detoxification of carcinogens in the liver and other tissues but is also a blood biomarker of hepatitis and liver cancer. Improved analytical methods are needed for the study of its role in the metabolism of xenobiotics and endogenous roles as a blood biomarker of diseases. The development of a double nanobody sandwich ELISA offers significant improvements over traditional polyclonal or monoclonal antibody-based assays, enhancing both the homogeneity and the stability of assay production. This study focuses on selecting and optimizing nanobody pairs for detecting human mEH. Four high-affinity nanobodies were identified and tested for thermal stability. Combinations of these nanobodies were evaluated, revealing that the MQ4-MQ30 pair achieved the best performance with a limit of detection (LOD) of 1 ng/mL. Additionally, polyHRP was also employed for signal amplification, enhancing detection capabilities despite challenges related to the small size and single epitope recognition of the nanobodies. Comparative studies using microplates and NHS@MF membranes were also performed. The superior performance of the NHS@MF membranes highlighted their potential as a promising alternative for point-of-care testing. The assay exhibited high specificity for human mEH and minimal cross-reactivity with related enzymes and effectively addressed matrix effects in plasma and tissue samples. These findings underscore the potential of double nanobody sandwich ELISAs for reliable and sensitive biomarker detection.

Determination of Trans-loxoprofen-alcohol Tromethamine in Pregnant SD Rats by a Validated LC-MS/MS Method：Application to a Toxicokinetics and Tissue Distribution Study

Background: As an anti-inflammatory prodrug, loxoprofen is metabolized into transloprofenol to treat diseases related to pain and inflammation. Although loxoprofen has fewer adverse effects than other NSAIDs, the safety of its usage during pregnancy remains unclear and needs to be considered. Fortunately, the toxicokinetics and tissue distribution study of transloxoprofen- alcohol in pregnant rats can resolve the problem. Objective: The purpose of this study is to establish a simple, sensitive, and effective LC-MS/MS analysis method for determining the concentration of trans-loxoprofen alcohol in plasma and tissues. Methods: The analytic samples were precipitated by methanol in one step and separated using a reverse-phase Poroshell 120 EC-C18 column (4.6 mm×50 mm; 2.7 μm). And the mobile gradient phase at a flow rate of 0.6 mL/min was composed of acetonitrile and 0.1% formic acid in water. The quantitative detection was achieved by multiple-reaction monitoring mode with a positive electrospray ionization source, transitional ion pairs of m/z 265.9>184.8 for trans-loxoprofenalcohol, and 268.8>187.9 for rac-trans-loxoprofen-D3 alcohol (Internal standard). Results: A good linearity of calibration curves for plasma and tissues was observed in the concentration range from 5.0 to 5000 ng/mL, and the lower limit of quantification was detected at 5.0 ng/mL. The intra-day and inter-day precision in plasma and tissues were within 8.94% and 7.26%, respectively. The mean extraction recovery and matrix effects in plasma and tissues were in the range of 89.08~109.27% and 89.00~106.80%, respectively. Precision of stability in plasma and tissues was within 8.91% and 7.08%, respectively. Conclusion: Complying with the requirements of bioanalytical guidelines by validation, this method was successfully adopted to the toxicokinetics and tissue distribution study after intravenously administrated trans-loxoprofen-alcohol into pregnant SD rats.

Alpha-Methylfentanyl and Beta-Hydroxyfentanyl LC-MS-MS Quantification in Rat Plasma after Long-Term Ethanol Exposure.

Fentanyl and its analogues are highly abused drugs that dominate the illicit drug trade. alpha-Methylfentanyl (A-F) and beta-hydroxyfentanyl (B-F) are two fentanyl analogues that require the development of rapid detection technologies. The current study established and validated a rapid and high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to measure A-F and B-F concentrations in rat plasma following intravenous drug administration (20 μg/kg). Because fentanyl is primarily metabolized by the liver, we evaluated the concentrations of A-F and B-F in vivo in rats, in a control group and a group with liver damage induced by 55 days of oral ethanol gavage (6.5 g/kg, 22.5% v/v). Liquid-liquid extraction and LC-MS-MS operating in the positive ion multiple reaction monitoring mode were used. A C18 column was used, and the mobile phase consisted of 0.1% formic acid aqueous and acetonitrile. The limit of detection was 3 pg/mL (S/N > 5) for A-F and B-F. The calibration curves were linear within the concentration range of 0.01-5 ng/mL (R2 = 0.9991) and 0.005-20 ng/mL (R2 = 0.9999) for A-F and B-F, respectively. Extraction recoveries were 91.3%-97.6% with RSD ≤ 11.2% and 90.5%-94.3% with RSD ≤ 10.5% for A-F and B-F, respectively. Plasma matrix effects were 80.61%-84.58% for A-F and 80.67%-81.33% for B-F with RSD ≤ 13.9%. The validated assay indicated no significant differences in pharmacokinetic parameters (AUC0-t, Cmax and T1/2) derived from the assessment of A-F and B-F plasma concentrations between control and ethanol-exposed rats. This assay, for which the LOD was 3 pg/mL for A-F and B-F may help the forensic science field to determine fentanyl analogue-related causes of death and identify illicit drug tampering.

Feasibility of Mass-Spectrometry to Lower Cost and Blood Volume Requirements for Assessment of B Vitamins in Patients Undergoing Bariatric Surgery.

Bariatric surgery induces deficiencies in a combination of B vitamins. However, high costs and a large blood volume requirement are barriers to routine screening. We adapted and validated a method coupling tandem mass spectrometry (MS/MS) with high-performance liquid chromatography (HPLC) to facilitate cost-effective analysis for simultaneous detection of B vitamins in low volumes of plasma. Based on existing methods, pooled plasma was extracted using hexane and acetonitrile and seven B vitamin analytes were separated using HPLC. Detection was performed with an Agilent 6460 triple quadrupole tandem mass spectrometer (MS/MS) using electrospray ionization in the positive ion mode. We evaluated linearity, recovery, precision, and limit of detection, as well as costs of the assay. We evaluated seven B vitamins from plasma; five (riboflavin, nicotinamide, pantothenic acid, pyridoxine, and biotin) were detected and quantified with precision and linearity. Recovery ranged from 63 to 81% for each of the vitamins, except for nicotinamide—the recovery of which was suppressed to 40%, due to plasma matrix effects. We demonstrated the feasibility of the HPLC–MS/MS method for use in patients who undergo bariatric surgery by analyzing pooled plasma from patients with a lower cost and blood volume than had we sent the samples to a commercial laboratory. It is advantageous and feasible, in terms of low cost and blood volume requirement, to simultaneously measure plasma concentrations of B vitamins using HPLC–MS/MS. With further improvements, the method may enable personalized nutritional assessment for the nutritionally compromised, bariatric surgery population.

Applicability of Supercritical fluid chromatography–Mass spectrometry to metabolomics. II–Assessment of a comprehensive library of metabolites and evaluation of biological matrices

In this work, the impact of biological matrices, such as plasma and urine, was evaluated under SFCHRMS in the field of metabolomics. For this purpose, a representative set of 49 metabolites were selected. The assessment of the matrix effects (ME), the impact of biological fluids on the quality of MS/MS spectra and the robustness of the SFCHRMS method were each taken into consideration. The results have highlighted a limited presence of ME in both plasma and urine, with 30% of the metabolites suffering from ME in plasma and 25% in urine, demonstrating a limited sensitivity loss in the presence of matrices. Subsequently, the MS/MS spectra evaluation was performed for further peak annotation. Their analyses have highlighted three different scenarios: 63% of the tested metabolites did not suffer from any interference regardless of the matrix; 21% were negatively impacted in only one matrix and the remaining 16% showed the presence of matrix-belonging compounds interfering in both urine and plasma. Finally, the assessment of retention times stability in the biological samples, has brought into evidence a remarkable robustness of the SFCHRMS method. Average RSD (%) values of retention times for spiked metabolites were equal or below 0.5%, in the two biological fluids over a period of three weeks.In the second part of the work, the evaluation of the Sigma Mass Spectrometry Metabolite Library of Standards containing 597 metabolites, under SFCHRMS conditions was performed. A total detectability of the commercial library up to 66% was reached. Among the families of detected metabolites, large percentages were met for some of them. Highly polar metabolites such as amino acids (87%), nucleosides (85%) and carbohydrates (71%) have demonstrated important success rates, equally for hydrophobic analytes such as steroids (78%) and lipids (71%). On the negative side, very poor performance was found for phosphorylated metabolites, namely phosphate-containing compounds (14%) and nucleotides (31%).

Ultrasensitive quantification of the CYP2E1 probe chlorzoxazone and its main metabolite 6-hydroxychlorzoxazone in human plasma using ultra performance liquid chromatography coupled to tandem mass spectrometry after chlorzoxazone microdosing.

Chlorzoxazone is a probe drug to assess cytochrome P450 (CYP) 2E1 activity (phenotyping). If the pharmacokinetics of the probe drug is linear, pharmacologically ineffective doses are sufficient for the purpose of phenotyping and adverse effects can thus be avoided. For this reason, we developed and validated an assay for the ultrasensitive quantification of chlorzoxazone and 6-hydroxychlorzoxazone in human plasma. Plasma (0.5mL) and liquid/liquid partitioning were used for sample preparation. Extraction recoveries ranged between 76 and 93% for both analytes. Extracts were separated within 3min on a Waters BEH C18 Shield 1.7μm UPLC column with a fast gradient consisting of aqueous formic acid and acetonitrile. Quantification was achieved using internal standards labeled with deuterium or (13)C and tandem mass spectrometry in the multiple reaction monitoring mode using negative electrospray ionization, which yielded lower limits of quantification of 2.5pgmL(-1), while maintaining a precision always below 15%. The calibrated concentration ranges were linear for both analytes (2.5-1000pgmL(-1)) with correlation coefficients of >0.99. Within-batch and batch-to-batch precision in the calibrated ranges for both analytes were <15% and <11% and plasma matrix effects always were below 50%. The assay was successfully applied to assess the pharmacokinetics of chlorzoxazone in two human volunteers after administration of single oral doses (2.5-5000μg). This ultrasensitive assay allowed the determination of chlorzoxazone pharmacokinetics for 8h after microdosing of 25μg chlorzoxazone.

Quantitative determination of mithramycin in human plasma by a novel, sensitive ultra-HPLC-MS/MS method for clinical pharmacokinetic application.

Mithramycin is a neoplastic antibiotic synthesized by various Streptomyces bacteria. It is under investigation as a chemotherapeutic treatment for a wide variety of cancers. Ongoing and forthcoming clinical trials will require pharmacokinetic analysis of mithramycin in humans, both to see if target concentrations are achieved and to optimize dosing and correlate outcomes (response/toxicity) with pharmacokinetics. Two published methods for mithramycin quantitation exist, but both are immunoassays that lack current bioanalytical standards of selectivity and sensitivity. To provide an upgraded and more widely applicable assay, a UPLC-MS/MS method for quantitation of mithramycin in human plasma was developed. Solid-phase extraction allowed for excellent recoveries (>90%) necessary for high throughput analyses on sensitive instrumentation. However, a ∼55% reduction in analyte signal was observed as a result of plasma matrix effects. Mithramycin and the internal standard chromomycin were separated on a Waters Acquity BEH C18 column (2.1×50 mm, 1.7 μm) and detected using electrospray ionization operated in the negative mode at mass transitions m/z 1083.5→268.9 and 1181.5→269.0, respectively, on an AB Sciex QTrap 5500. The assay range was 0.5-500 ng/mL and proved to be linear (r(2)>0.996), accurate (≤10% deviation), and precise (CV<15%). Mithramycin was stable in plasma at room temperature for 24 h, as well as through three freeze-thaw cycles. This method was subsequently used to quantitate mithramycin plasma concentrations from patients enrolled on two clinical trials at the NCI.

The mass transfer dynamics of hollow fiber liquid-phase microextraction and its application for rapid analysis of biological samples

Hollow fiber liquid-phase microextraction (HF-LPME) has been demonstrated to potentially become a mainstream sample preparation technique for complex samples. Nevertheless, the need for a relatively long extraction time is considered to be the major disadvantage of this method. Lengthy extractions may cause the loss of the extraction phase and may change the contents of biological samples via the action of enzymes. Therefore, control calibrations for particular biological systems must be made. In this study, a theoretical model of the mass transfer dynamics of two-phase HF-LPME was proposed, and the kinetic calibration (KC) of this method for plasma and urine samples was validated. The theoretical results were validated by examining the kinetics of the extraction and back-extraction processes of HF-LPME. The KC-HF-LPME method was successfully used to correct for matrix effects in plasma and urine samples during flunitrazepam analysis. The free amount of flunitrazepam was extracted from plasma for 10min and analyzed by gas chromatography/mass spectrometry. The amount of pre-added standard and the standard remaining in the extraction phase after extraction were used for the quantification of flunitrazepam in plasma and urine samples. The new method not only significantly shortens the extraction time but also provides a new opportunity to determine the free concentration of analyte in biological systems.

Quantification of femtomolar concentrations of the CYP3A substrate midazolam and its main metabolite 1′-hydroxymidazolam in human plasma using ultra performance liquid chromatography coupled to tandem mass spectrometry

The benzodiazepine midazolam is a probe drug used to phenotype cytochrome P450 3A activity. In this situation, effective sedative concentrations are neither needed nor desired, and in fact the use of very low doses is advantageous. We therefore developed and validated an assay for the femtomolar quantification of midazolam and 1'-hydroxymidazolam in human plasma. Plasma (0.25 mL) and 96-well-based solid-phase extraction were used for sample preparation. Extraction recoveries ranged between 75 and 92% for both analytes. Extracts were chromatographed within 2 min on a Waters BEH C18 1.7 μm UPLC® column with a fast gradient consisting of formic acid, ammonia, and acetonitrile. Midazolam and 1'-hydroxymidazolam were quantified using deuterium- and (13)C-labeled internal standards and positive electrospray tandem mass spectrometry in the multiple reaction monitoring mode, which yielded lower limits of quantification of 50 fg/mL (154 fmol/L) and 250 fg/mL (733 fmol/L) and a corresponding precision of <20%. The calibrated concentration ranges were linear for midazolam (0.05-250 pg/mL) and 1'-hydroxymidazolam (0.25-125 pg/mL), with correlation coefficients of >0.99. Within-batch and batch-to-batch precision in the calibrated ranges for both analytes were <14% and <12%. No ion suppression was detectable, and plasma matrix effects were minimized to <15% (<25%) for midazolam (1'-hydroxymidazolam). The assay was successfully applied to assess the kinetics of midazolam in two human volunteers after the administration of single oral microgram doses (1-100 μg). This ultrasensitive assay allowed us to quantify the kinetics of midazolam and 1'-hydroxymidazolam for at least 10 h, even after the administration of only 1 μg of midazolam.

A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.

Quantitative analysis of carbonaceous aerosols using laser-induced breakdown spectroscopy: a study on mass loading induced plasma matrix effects

We present results indicating mass loading induced plasma matrix effects on the application of quantitative laser-induced breakdown spectroscopy (LIBS) for estimation of carbon contents in aerosols. An in-house flow-controlled powder-dispersion system generated carbonaceous aerosols with varying bi-modal particle size distributions (∼1 µm and 10 µm median diameters), thereby resulting in a wide mass loading range. For ease of chemical handling and to eliminate toxic effects, common talcum powder was used as our standard aerosol. Normalized atomic species concentrations of C, i.e., [C]/[Si] ratios, were calculated from atomic emission lines of C I (248 nm), Si I (252 nm), and plasma temperatures estimated from a series of Mg I lines. The results show a decrease in [C]/[Si] ratio to about 65% of the initial value as relative mass loadings increased (5.5–100%) due to the increase in number concentrations of larger sized particles (∼10 µm median diameter). As a comparison, normalized ratio of [Mg]/[Si] did not exhibit any marked change with increased mass loading. The normalized total absorption of photon flux across the C I (248 nm) spectral line indicated a strong correlation to the percentage decrease in [C]/[Si] ratio. We used an impactor with a cut-off size of around 10 µm diameter to generate mono-modal aerosolized powders (∼1 µm median diameter) that had lower relative mass loadings (0.32–0.16%). Similar LIBS analysis on these did not indicate any of the matrix effects. We conclude that for aerosol systems with widely varying mass loadings, quantitative LIBS analysis can be significantly affected by plasma matrix effects, specifically for the C I (248 nm) emission line as noticed in this study. This bears significance for the application of quantitative LIBS in the chemical characterization of all forms of carbonaceous aerosols.

Determination of rifalazil, a potent antibacterial agent, in human plasma by liquid–liquid extraction and LC–MS/MS

A sensitive assay for determination of rifalazil (also known as ABI-1648 and KRM-1648) in human plasma is described. The analytical method utilizes liquid–liquid extraction of plasma with methyl tert-butyl ether, followed by reversed-phase liquid chromatography with a C 18 column and a mobile phase gradient utilizing 0.1% formic acid in water and acetonitrile, respectively. Electrospray mass spectrometry in the positive ion mode with selected reaction monitoring of rifalazil and an isotope labeled internal standard, 13C 4-rifalazil (ABI-9901) was used for selective and sensitive detection. The calibration range was 0.050–50 ng/mL plasma using 200 μL plasma sample volume. The absolute extraction recovery of rifalazil from K 2-EDTA plasma, evaluated at three concentration levels, was 88.6–97.3%, and the recovery for the internal standard was 96.8%. A study of plasma matrix effects showed a peak area response at 90–99% compared to neat solutions for both rifalazil and the internal standard. Stability evaluation of rifalazil in plasma, whole blood and methanol showed that the analyte stability was adequate when stored under study conditions. The precision, as evaluated in three validation batches, was consistent for fortified plasma quality control (QC) samples at four concentration levels, with ≤6% R.S.D. except for at the lowest quality control level where it was 10.7% R.S.D. The accuracy for QC samples (difference between found and nominal concentration) ranged from −2.3% to 5.1%. Similar precision and accuracy values were obtained over 6 months of routine application of this method. It was concluded that the performance improved markedly during routine operation by replacing a closely related structural analog internal standard with the stable isotope internal standard.

Quantitative analysis of alloys and glasses by a calibration-free method using laser-induced breakdown spectroscopy

Space-, time- and spectrally resolved optical diagnostics of laser ablation plasma has provided the opportunity to realize calibration-free analyses of solid materials. In general, this variant of optical emission spectroscopy of pulsed plasma allows the plasma matrix effects to be overcome, yielding satisfactorily precise and accurate quantitative results on elemental composition of materials without using calibration curves, certified reference materials, and internal standards. Such analysis is very close to be nondestructive due to the minimum possible ablated mass, a feature which is very important in many applications, especially for unique museum exhibits and jeweler samples. In this paper, the use of the method for the analysis of elements in bronze, brass and gold alloys, glass samples, and archaeological findings is demonstrated. The results presented confirm the suitability of the approach for routine applications of our instrumentation, while at the same time simplifying the overall analytical procedure.

Simultaneous quantitative determination of deuterium- and carbon-13-labeled essential fatty acids in rat plasma

This study reports methods for the quantitative determination of stable isotope-labeled essential fatty acids (EFAs) as well as an experiment in which deuterium-labeled linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) were compared with those labeled with carbon-13 in rat plasma in vivo. Standard curves were constructed to compensate for concentration and plasma matrix effects. It was observed that endogenous pools of fatty acids had a greater suppressing effect on the measurements of 13C-U-labeled EFAs relative to those labeled with 2H5. Using these methods, the in vivo metabolism of orally administered deuterated-linolenate, 13C-U-labeled linolenate, deuterated-linoleate, and 13C-U-labeled linoleate was compared in adult rats (n = 11). There were no significant differences in the concentrations of the 2H versus 13C isotopomers of 18:2n-6, 18:3n-3, arachidonic acid (20:4n-6), and docosahexaenoic acid (22:6n-3) in rat plasma samples at 24 h after dosing. Thus, there appears to be little isotope effect for 2H5- versus 13C-U-labeled EFAs when the data are calculated using the conventional standard curves and corrected for endogenous fatty acid pool size and matrix effects.

A predictive model of plasma matrix effects in inductively coupled plasma atomic emission spectrometry

Download options Please wait... Article information DOI https://doi.org/10.1039/JA9860100185 Article type Paper Download Citation J. Anal. At. Spectrom., 1986,1, 185-193 BibTex EndNote MEDLINE ProCite ReferenceManager RefWorks RIS Permissions Request permissions A predictive model of plasma matrix effects in inductively coupled plasma atomic emission spectrometry M. H. Ramsey and M. Thompson, J. Anal. At. Spectrom., 1986, 1, 185 DOI: 10.1039/JA9860100185 To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page. If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given. If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page. Read more about how to correctly acknowledge RSC content. Social activity Tweet Share Search articles by author Michael H. Ramsey Michael Thompson

Determination of copper in glasses by flame atomic absorption and plasma emission spectrometry

Ground samples (150μm, 0.5 g) are decomposed in a mixture of HF and HNO3 acids followed by evaporation to dryness, dissolution of the residue in dilute HNO3 acid, and dilution to 50 ml. Spectral interferences of 14 tested elements are negligible for AAS but in some cases (Nb, Ti, Zr) minor spectral corrections must be made for plasma techniques depending on the level of interferent and analyte. Nonspectral (matrix) interferences at the 1000μg/ml interferent level are all less than 3.5% relative for AAS and less than 5% relative for ICP (except for Ca, Li, and Na where the effects are depressions of 7 to 10% relative). Matrix effects for DCP at the same interferent level are generally more pronounced and amount to enhancements of 5 to 15% relative for 11 of the 14 elements tested. Plasma matrix effects, especially alkali effects, can be compensated by the use of an alkali-aluminum buffer medium (1000μg Na/ml plus 500μg Al/ml as used in this study), a procedure which is most useful for the DCP approach. Copper can be determined down to 10 ppm in the original sample. A comparison of results shows AAS and ICP to be in good agreement with the DCP being slightly less accurate, although the differences in results may be of little practical significance. In terms of freedom from spectral and non-spectral interference, AAS was judged to have a slight advantage for copper.