Matrix Suppression Research Articles

A Modified QuEChERS Extraction and LC-MS/MS Method for the Determination of Pesticide Residues in Curry Leaves (Murraya koenigii)

Background:Increased consumer demand for curry leaves free from pesticides demands fast and reliable analytical methods for the analysis of pesticide residues.Objective:The optimization of a QuEChERS based sample preparation technique with improved analytical accuracy by removing interfering matrix components for LC-MS/MS analysis of pesticide residues from curry leaves.Methods:A modified QuEChERS solid phase extraction method was developed and validated for the analysis of 26 pesticides in fresh and dried curry leaves. The effects of the sample preparation steps and column retention time on the matrix suppression of analyte ions were also evaluated.Results:Validation parameters were found within an acceptable range. The matrix effect evaluation studies showed that the QuEChERS sample preparation was able to minimize the ion suppression of analytes due to co-eluting matrix of components and that a d-SPE clean up step had major role in reducing matrix effect. The gradient mobile phase with longer retention time for analytes resulted in comparatively lesser matrix effects than the isocratic mobile phase of non-polar nature. Even after the clean up, a considerable number of compounds had more than 20% reduction in their MS response in the gradient mobile phase.Conclusion:This study emphasized the need of proper sample clean up before a LC-MS/MS analysis and the usage of matrix matched standards and mobile phase that ultimately results in an appropriate analyte separation in reasonable retention times.

Determination of Nitrofuran Metabolites in Muscular Tissue by High-Performance Liquid Chromatography with Mass Spectrometric Detection

We developed a procedure for determining residual amounts of four nitrofuran metabolites (3‑amino-2-oxazolidinone, 3-amino-5-methylmorpholino-2-oxazolidinone, 1-aminohydantoin hydrochloride, and semicarbazide hydrochloride) in muscle tissue in the form of 2-nitrophenyl derivatives by HPLC with selected reaction monitoring mass spectrometric detection. The effect of the matrix suppression of ionization is shown. The selectivity of extraction is increased by optimizing the sample preparation procedure, and the effect of organic impurities on the result measurements is minimized. The lower limit of the analytical range is 10 μg/kg for semicarbazide hydrochloride and 1 μg/kg for the remaining nitrofuran metabolites.

Matrix interference in LC-ESI-MS/MS analysis of metanephrines in protein precipitated plasma samples

Metanephrine and normetanephrine are measured in blood plasma to diagnose different diseases. Simpler sample preparation procedures are preferred but tend to yield less purified extracts. Therefore, thorough investigation of matrix effects is required. In this work, several sample preparation methods and chromatographic modes were compared for liquid chromatography tandem mass spectrometric (with electrospray ionization; LC-ESI-MS/MS) analysis of metanephrine and normetanephrine in blood plasma. Protein precipitation with methanol was found to be sufficient for sample preparation and pentafluorophenyl column provided adequate chromatographic separation. A new cheaper and less labor-intensive approach is proposed where necessary quantitation limits are achieved through a sample preparation containing only protein precipitation and dilution of the sample extract. Matrix effects for different sample preparation methods and the use of isotope-labeled internal standards were evaluated. Unusual interference to D3-labeled internal standard of normetanephrine was discovered - signal of interfering compound increased while the matrix effects were reduced by dilution, e.g. dilution eliminates matrix suppression on interfering compound. The results stress the need to monitor interfering compounds and evaluate matrix effects at every step of method development. Matrix effects and interferences can be different for analytes and their corresponding isotopically labeled internal standards. This means that the use of isotopically labeled internal standards cannot guarantee accuracy of obtained results. New method allows quantification of the low nanomolar concentrations of metanephrine and normetanephrine in plasma samples.

Detection and quantification of metastable photoproducts of trenbolone and altrenogest using liquid chromatography–tandem mass spectrometry

Here, we developed a novel and sensitive method for the detection and quantification of metastable trenbolone and altrenogest photoproducts in agricultural receiving waters based on solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC–MS/MS). Primary method analytes were seven cycloaddition or photohydration transformation products of 17α-trenbolone (17α-TBOH), 17β-trenbolone (17β-TBOH), trendione (TBO), and altrenogest (ALT), which are key contributors to the fate and environmental risks of these steroidal pharmaceuticals. Because commercial analytical standards are not available, reference standards for photoproducts were generated from trenbolone or ALT with a solar simulator (˜6 h, >10 half-lives). Efficient detection of metastable photoproducts required cold and pH neutral conditions, rapid sample processing, minimal sample storage, and consideration of cationic artifacts. Method detection limits (MDLs) were 0.034-0.40 ng L−1 for parent compounds and 0.16–2.1 ng L−1 for photoproducts, sufficient for their detection in agroecosystems. Matrix suppression was observed and corrected by internal standards, and relative recovery rates were near 100% for all analytes except for 12-OH-17α-TBOH (˜75% recovery). Intra-day variation was <20% and inter-day variation <25% for all the analytes. The developed method is capable of the analysis of trenbolone, altrenogest, and their key bioactive photoproducts in agricultural receiving waters.

Runx1 contributes to articular cartilage maintenance by enhancement of cartilage matrix production and suppression of hypertrophic differentiation

Osteoarthritis (OA) results from an imbalance of the dynamic equilibrium between the breakdown and repair of joint tissues. Previously, we reported that Runx1 enhanced chondrogenic differentiation through transcriptional induction of COL2A1, and suppressed hypertrophic differentiation. Here, we investigated the involvement of Runx1 in OA development as well as its potential underlying molecular mechanism. When we analysed OA development in Col2a1-Cre;Runx1fl/fl and Runx1fl/fl mice by surgically inducing joint instability, Cartilage degradation and osteophyte formation of Col2a1-Cre;Runx1fl/fl joints was accelerated compared with joints in Runx1fl/fl animals 8 weeks after surgery. To investigate chondrocyte regulation by Runx1, we analysed interactions with co-factors and downstream molecules. Runx1 enhanced cartilage matrix production in cooperation with Sox5, Sox6, and Sox9, and co-immunoprecipitation assays showed protein–protein binding between Runx1 and each Sox protein. Knockdown of Runx1 increased expression of a hypertrophic marker, Co10a1, in mouse articular cartilage and primary chondrocytes. This expression was accompanied by decreased expression of Bapx1, a potent suppressor of hypertrophic differentiation. Notably, Runx1-induced suppression of hypertrophic differentiation was diminished by siRNA silencing of Bapx1, whereas chondrogenic markers were unaltered. Thus, Runx1 contributes to articular cartilage maintenance by enhancing matrix production in cooperation with Sox proteins, and suppressing hypertrophic differentiation at least partly via Bapx1 induction.

Determination of microcystins, nodularin, anatoxin-a, cylindrospermopsin, and saxitoxin in water and fish tissue using isotope dilution liquid chromatography tandem mass spectrometry

Cyanobacteria can form dense blooms under specific environmental conditions, and some species produce secondary metabolites known as cyanotoxins, which present significant risks to public health and the environment. Identifying toxins produced by cyanobacteria present in surface water and fish is critical to ensuring high quality food and water for consumption, and protectionn of recreational uses. Current analytical screening methods typically focus on one class of cyanotoxins in a single matrix and rarely include saxitoxin. Thus, a cross-class screening method for microcystins, nodularin, anatoxin-a, cylindrospermopsin, and saxitoxin was developed to examine target analytes in environmental water and fish tissue. This was done, due to the broad range of cyanotoxin physicochemical properties, by pairing two extraction and separation techniques to improve isolation and detection. For the first time a zwitterionic hydrophilic interaction liquid chromatography column was evaluated to separate anatoxin-a, cylindrospermopsin, and saxitoxin, demonstrating greater sensitivity for all three compounds over previous techniques. Further, the method for microcystins, nodularin, anatoxin-a, and cylindrospermopsin were validated using isotopically labeled internal standards, again for the first time, resulting in improved compensation for recovery bias and matrix suppression. Optimized extractions for water and fish tissue can be extended to other congeners in the future. These improved separation and isotope dilution techniques are a launching point for more complex, non-targeted analyses, with preliminary targeted screening.

Integration of stable isotope labeling derivatization and magnetic dispersive solid phase extraction for measurement of neurosteroids by in vivo microdialysis and UHPLC-MS/MS

In this work, a novel strategy of stable isotope labeling derivatization (SILD) combined with magnetic dispersive solid-phase extraction (MDSPE), has been proposed for simultaneous monitoring of neurosteroids changes linked to Parkinson's disease (PD) by in vivo microdialysis. The developed method was based on ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection using multiple-reaction monitoring (MRM) mode. In this study, a new pair of stable isotope labeling reagents d0-/d3-3-N-methyl-2′-carboxyl Rhodamine 6G (d0-/d3-MCR6G), were designed and synthesized for derivatizing neurosteroids in rat blood microdialysates and neurosteroid standards, respectively. d3-MCR6G labeled neurosteroids standards were used as internal standard for the following pretreatment and UHPLC-MS/MS quantification to minimize the deviations caused by complex matrix and ion suppression effects in mass spectrometry analysis. Under the optimized derivatization and extraction conditions, good linearities of eight neurosteroids were obtained with correlation coefficients R2 values > 0.98. The limits of detection (LODs) and quantitation (LOQs) ranged from 0.06 to 0.12 pg/mL and 0.30–0.40 pg/mL, respectively. Taken together, the established method exhibited high sensitivity and selectivity, excellent accuracy, convenience and high efficiency. It was applied for the simultaneous and dynamic measurement of multiple neurosteroids in normal and PD rat blood microdialysates. This method would be expected to be potentially useful for the monitoring and drug treatment of PD and related neurological disorders in the future.

A sensitive and robust UPLC–MS/MS method for quantitation of estrogens and progestogens in human serum

ObjectiveWith the widespread use of sex-steroid hormones in contraceptives and hormone replacement therapy, there is an increasing need for reliable analytical methods. We report the development of a sensitive and robust UPLC–MS/MS method for quantitation of both endogenous and synthetic sex-steroid hormones in human serum. Study designWe developed and validated a UPLC–MS/MS method to quantify progestogens (etonogestrel, levonorgestrel, medroxyprogesterone acetate, norethindrone, progesterone) and estrogens (estradiol and ethinyl estradiol) with good accuracy, high sensitivity, and excellent robustness. We then applied the method to the analysis of sex-steroid hormones in serum from 451 clinical research participants. ResultsEach UPLC–MS/MS analysis was 6.5 min. The lower limits of quantitation (LLOQs) were 25 pg/ml for the progestogens, and 2.5 and 5.0 pg/ml for estradiol and ethinyl estradiol, respectively. When estradiol was analyzed without assessment of progestogens, the LLOQ was reduced to 1 pg/ml. The calibration curves were linear from 25–50,000, 2.5–2000 (1–2000 for estrogens-only analysis) and 5–2000 pg/ml, respectively. Both the accuracy and precision were below±15% not only for routine validation (intraday and interday), but for long-term (>2 years) assay robustness with external controls, thereby, demonstrating the utility of this method for multi-year clinical trial assessments of progestogens and estrogens. We applied the method to quantify sex-steroid levels in 1804 clinical samples. ConclusionsWe successfully developed a UPLC-MS/MS method, and overcame the matrix suppression to allow sensitive quantitation of both synthetic and endogenous sex-steroid hormones in human serum. ImplicationsWe developed a sensitive and robust UPLC-MS/MS method to accurately measure the levels of sex-steroid hormones in serum. The method overcame matrix interference barriers and achieved excellent long-term stability and reproducibility (≥96.9% accuracy; ≤13.0% relative variability measured with external controls over 2 years), demonstrating its utility in clinical sample analysis.

Pyrethroids residues analysis in Indonesian commercial tea by GC-ECD

Monitoring of pesticides residue including pyrethroids is required since they are major contaminants in tea and have been regulated in European Union and countries such as China, Australia, Japan and America. This study aimed to analyze several pyrethroids in Indonesian commercial tea. The analysis was done by applying low volume solid-liquid extraction followed by quantification by gas chromatography-electron capture detector as a relatively cheaper alternative instrument in pesticides monitoring compared to mass spectrophotometer. The detection limits of the method were 0.006 mg/kg for deltamethrin, 0.05 mg/kg for fenvalerate, 0.10 mg/kg for cypermethrin and 0.05 mg/kg for λ-cyhalothrin. The linearity of the method was good with coefficient correlation of higher than 0.997 at 0.3 to 2.0 mg/kg. The repeatability of the method was also good with lower % relative standard deviations compared to two-thirds of Horwitz coefficient of variation and European Commission guideline. The recoveries were all in the range suggested by European Commission. Weak matrix effect was observed for deltamethrin and cypermethrin while matrix suppression effect was observed for λ-cyhalothrin and fenvalerate. Analysis of tea samples planted and produced in Indonesia showed that the pyrethroid residue in the samples were all below the maximum residue limits while the risk assessment suggested that Indonesian commercial tea is safe for consumption.

High-Throughput Quantitation of Neonicotinoids in Lyophilized Surface Water by LC-APCI-MS/MS.

Background: Neonicotinoids are among the most widely used insecticides. Recently, there has been concern associated with unintended adverse effects on honeybees and aquatic invertebrates at low parts-per-trillion levels. Objective: There is a need for LC-MS/MS methods that are capable of high-throughput measurements of the most widely used neonicotinoids at environmentally relevant concentrations in surface water. Methods: This method allows for quantitation of acetamiprid, clothianidin, imidacloprid, dinotefuran, nitenpyram, thiacloprid, and thiamethoxam in surface water. Deuterated internal standards are added to 20 mL environmental samples, which are concentrated by lyophilisation and reconstituted with methanol followed by acetonitrile. Results: A large variation of mean recovery efficiencies across five different surface water sampling sites within this study was observed, ranging from 45 to 74%. This demonstrated the need for labelled internal standards to compensate for these differences. Atmospheric pressure chemical ionization (APCI) performed better than electrospray ionization (ESI) with limited matrix suppression, achieving 71-110% of the laboratory fortified blank signal. Neonicotinoids were resolved on a C18 column using a 5 min LC method, in which MQL ranged between 0.93 and 4.88 ng/L. Conclusions: This method enables cost effective, accurate, and reproducible monitoring of these pesticides in the aquatic environment. Highlights: Lyophilization is used for high throughput concentration of neonicotinoids in surface water. Variations in matrix effects between samples was greatly reduced by using APCI compared with ESI. Clothianidin and thiamethoxam were detected in all samples with levels ranging from below method quantitation limit to 65 ng/L.

Determination of thiol-containing drugs in human plasma by stable isotope labeling coupled with high performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis

A pair of stable isotope labeling (SIL) reagents, N-(4-(carbazole-9-yl)-phenyl)-N-maleimide (NCPM-d0) and its heavy analogue NCPM-d2, were used for labeling thiol-containing drugs. On basis of SIL, a global isotope internal standard quantitative method for the detection of five thiol-containing drugs by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. The NCPM-d0 and NCPM-d2 can easily label thiol-containing drugs under mild conditions within 10 min at 40 °C. The NCPM-d0 and NCPM-d2 labeled thiol-containing drugs can generate two characteristic product ions (m/z at 372.5 and 374.5) under collision induced dissociation, respectively, which is used to establish the multiple reaction monitoring (MRM) based detection. The NCPM labeling combined with MRM analysis not only allowed trace detection of thiol-containing drugs due to the extremely high sensitivity, but also efficiently corrected the matrix effects during HPLC-MS/MS and the instrument fluctuation in the MS/MS signal intensity. The detection sensitivities of thiol-containing drugs improved by 14.5–650.5-fold due to NCPM-labeling, while the matrix and ion suppression effects were markedly minimized by the SIL strategy. The limits of detection (LODs) and the limits of quantitation (LOQs) were in the range 10.0–15.0 ng·mL−1 and 31.0–50.0 ng·mL−1, respectively. The proposed method was used for the simultaneous determination of five thiol-containing drugs in plasma samples with satisfactory recoveries in the range of 95.0–97.5%.

Abstract NG04: Immune evasion mechanisms in melanoma resistant to targeted- and/or immunotherapy

Abstract NG04: Immune evasion mechanisms in melanoma resistant to targeted- and/or immunotherapy

Rapid determination of polar pesticides and plant growth regulators in fruits and vegetables by liquid chromatography/tandem mass spectrometry

ABSTRACTA simple high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) multiresidue analysis method was developed for the simultaneous determination of polar pesticides, plant growth regulators and other polar compounds. These included amitrole, chlormequat, mepiquat, cyromazine, ETU, PTU, perchlorate, and daminozide using a mixed-mode column. A 10 g test portion was shaken with acidified methanol for 10 min. After centrifugation, the sample extract was injected and analyzed within 11 min by LC-MS-MS. This column eliminated the need for derivatization or the use of ion paring reagent. Two MS-MS transitions were monitored in the method for each target compound to achieve true positive identification. Eight isotopically-labeled internal standards corresponding to each analyte were used to correct for matrix suppression effect and/or instrument signal drift. The average recovery for all analytes at 20, 40, and 250 ng/g (n = 6) ranged from 73–136%, with a relative standard deviation of ≤ 20%.

Two-stage probe atomization GFAAS for direct determination of trace Cd and Pb in whole bovine blood

Graphite furnace atomic absorption spectrometry (GFAAS) is rarely used for direct trace analysis of whole blood because of strong interferences caused by the organic matrix. Especially it concerns bovine blood samples, containing more proteinaceous and lipid components in comparison with human blood. In our work, the novel two-stage probe atomization (TPA) technique for Cd and Pb determination by GFAAS in whole bovine blood with minimum sample pre-treatment has been developed. Making use of TPA and chemical modifier has eliminated matrix suppression and decreased the non-selective absorption level by several times. It allowed us to dispense with brief sample pre-treatment by a mixture of nitric acid and hydrogen peroxide (1:1) without autoclave digestion. The pyrolysis temperatures were 650 °C for Cd and 900 °C for Pb in the presence of Pd modifier. The analytical signal dependence on the number of concentrating cycles has been studied. The maximal number of capturing cycles was found (15 and 10 for Cd and Pb). Applying of multiple probe concentrating has additionally decreased the limits of detection (LODs) comparing with single TPA. The achievable LODs in optimized conditions were found to be 0.01 and 0.2 μg/L for Cd and Pb, respectively (3 s test). The accuracy of the proposed technique was confirmed by anodic stripping voltammetry (ASV). The relative standard deviation for 5.1 μg/L Cd and 26 μg/L Pb concentrations was 6.4% and 7.5%, respectively.

Anti-invasion and anti-metastasis effects of Valjatrate E via reduction of matrix metalloproteinases expression and suppression of MAPK/ERK signaling pathway

Valjatrate E is an iridoid compound extracted from Valeriana jatamansi Jones herb and is the active ingredient in antitumor activity. Here, we reported its action on tumor invasion and metastasis in the human hepatocellular carcinoma HepG2, aiming at a better understanding of the potential mechanism of action of Valjatrate E. HepG2 cells were treated with Valjatrate E at different concentrations. Wound healing assay and transwell chamber assay were used to determine the effects of Valjatrate E on the migration and invasiveness of HepG2 cells, respectively. Moreover, homogeneity and heterotypic adhesion experiments evaluated the adhesion property of HepG2 cells. The molecular mechanisms by which Valjatrate E inhibited the invasion and migration of HepG2 cells were investigated by gelatin zymography experiment and western blot. Treatment with Valjatrate E inhibited the migration and invasion of HepG2 cells. It achieved this by reducing the expression of matrix metalloprotease 2 (MMP-2) and matrix metalloprotease 9 (MMP-9), by inhibition of heterogeneous adhesion ability, by blocking mitogen-activated protein kinase (MAPK) signaling via inhibiting the phosphorylation of extracellular signal-regulated kinases (p-ERK). Taken together, these findings provide new evidence that mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) signaling pathway plays an important role in promoting invasion and metastasis in HepG2 cells through p-ERK, and MAPK/ERK signaling pathway may be a therapeutic target for tumor.

Tracing the Origin of the Anomalous Matrix Suppression in Matrix‐assisted Laser Desorption Ionization and Extending the Linear Dynamic Range During Analyte Quantification

In the matrix‐assisted laser desorption ionization‐based quantification method developed in this laboratory, a linear calibration curve is constructed by plotting the analyte‐to‐matrix ion abundance ratio as a function of the analyte concentration. As the analyte concentration increases, however, the curve deviates from linearity. Previously, we observed that the occurrence of this deviation correlated with the decrease in the matrix ion signal, or matrix suppression. In this work, we found that saturation of the detector was one of the factors responsible for the above phenomena. As we decreased the detector gain, the upper limit of the linear dynamic range increased by as much as one order of magnitude. Even after a substantial decrease in the detector gain, however, the nonlinearity remained at a high analyte concentration, which we cannot account for at present. The method developed in this work can be useful for the quantitative imaging of analytes in heavily contaminated samples.

Drug Monitoring by Volumetric Absorptive Microsampling: Method Development Considerations to Mitigate Hematocrit Effects

GSKA is a compound that was in development in clinical trials. A bioanalysis method to quantify GSKA using volumetric absorptive microsampling (VAMS)was developed and hematocrit (HCT) related assay bias was investigated. After accurate sampling of 10μl blood, VAMS tips were air dried approximately 18h and desorbed by an aqueous solution containing internal standard. The recovered blood underwent liquid-liquid extraction in ethyl acetate to minimize matrix suppression. Assay accuracy, precision, linearity, carryover, selectivity, recovery, matrix effects, HCT effects and long-term quality control stability were evaluated. HCT-related assay bias was minimized in 30-60% blood HCT range, and all validation parameters met acceptance criteria. The method is suitable for quantitative analysis of GSKA in human blood.

Detection of cathinone and mephedrone in plasma by LC-MS/MS using standard addition quantification technique

BackgroundCathinones better known as bath salts have been listed as illicit drugs since 2011. Few studies have focused on the analytical extraction techniques and the matrix effect affecting their detection and quantification in biological samples. Matrix suppression of the signal of cathinones has been previously observed in urine sample by LC-MS/MS. This study is aimed to use the standard addition method to overcome the plasma matrix effect on the quantification of cathinone and mephedrone by LC-MS/MS.FindingsThe results showed the matrix effect for cathinone at lowest tested concentration (10 ng/ml) was significantly reduced from 210.9 to 133.7%, but not for mephedrone (from 196.8 to 191.9%) by using standard addition method. At the higher tested concentrations of samples, the matrix effects were significantly reduced for both cathinone and mephedrone by using standard addition technique.ConclusionsStandard addition quantitative technique can serve as an alternative quantitative method for LC-MS/MS when suitable internal standards are not available.

Determination of Dinotefuran and Its Metabolites in Orange Pulp, Orange Peel, and Whole Orange Using Liquid Chromatography-Tandem Mass Spectrometry.

Determination of the polar characteristics of dinotefuran and metabolite residues in orange matrixes (orange pulp, orange peel, and whole orange) is difficult. Thus, the purpose of the present study was to develop an extraction method for the determination of dinotefuran and its metabolites in oranges by using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Matrix suppression effects were observed for all analytes in the orange matrixes. The proposed method displayed satisfactory linearity (R2 ≥ 0.9856) for the target molecules. The LODs were 0.03-0.10 mg/kg, whereas LOQs were 0.08-0.40 mg/kg for dinotefuran and its metabolites. Recoveries were 79.1-98.7% with RSD values <20% for all analytes in the orange matrixes. The proposed method was used to authenticate the samples and dinotefuran residues observed in field-incurred orange matrixes. The results of the proposed method could help the Chinese government establish maximum residue limits for dinotefuran in oranges and promote the safe and proper use of dinotefuran dosages in orange trees.

Quantification of Loperamide by Gas Chromatography Mass Spectrometry

Due to reported pharmacological activity similar to classical opioids at supratherapeutic concentrations, abuse of the anti-diarrheal medication loperamide (Imodium AD™) has become a target in the opioid epidemic. While this phenomenon is not new, published quantitative analytical methods use liquid chromatography tandem mass spectrometry. Described here is an 11 min method for quantification of loperamide in postmortem whole blood by gas chromatography mass spectrometry. Validation studies performed followed SWGTOX guidelines and included: accuracy, specificity, limit of detection (LOD), regression model analysis, stability, and matrix recovery enhancement and/or suppression. The accuracy study consisted of inter-day, intra-day, reproducibility and dilution integrity experiments. Inter-day and intra-day accuracy, precision and coefficient of variation (CV) were measured; normalized results were 1.05 ± 0.09 with 8.87% CV (n = 36) and 1.03 ± 0.09 with 8.53% CV (n = 27), respectively. Reproducibility was evaluated through standard addition with an observed CV of 10.84% (n = 10). Dilution integrity (2× and 4×) resulted in 0.94 ± 0.13 with a CV of 13.9% (n = 5). No interference was observed through analyses of the internal standard (loperamide-d6), endogenous compounds (10 blank matrices) or 60 commonly encountered analytes. The LOD/decision point was 100 ng/mL (CV 8.40%). A linear calibration model was established from 100 to 1,000 ng/mL. Stability was examined; observed analyte-to-internal standard response resulted in 6.59% CV. Recovery was determined for loperamide and loperamide-d6 (31% and 36%, respectively). Neither matrix suppression nor enhancement was observed with loperamide at 750 ng/mL and loperamide-d6 at 300 ng/mL (-6.5% and -4.2%); however, some suppression was exhibited at lower concentrations (-39.8%). The designed method was determined to be sufficient for the analysis of loperamide-related death cases in Alabama (n = 8) and offers postmortem toxicology laboratories an alternative approach that is both highly selective and specific.