Sea urchin nanostructured nickel cobaltite modified carbon cloth integrated wearable patches for the on-site detection of the immunosuppressant drug mycophenolate mofetil.

Mycophenolate mofetil (MpM) is a medication used to prevent the rejection of transplanted organs, particularly in kidney, heart, and liver transplant surgeries. It is extremely important to be conscious that MpM can raise the risk of severe infections and some cancers if it exceeds the recommended dose while lower doses will result in organ rejections. So, it is essential to monitor the dosage of MpM in real time in the micromolar range. In this work, we have synthesized 3-aminopropyltriethoxysilane (APTES) functionalized nickel cobaltite (NiCo2O4) and this amino functionalization was chosen to enhance the stability and electrochemical activity of NiCo2O4. The enhanced activity of NiCo2O4 was used for developing an electrochemical sensor for the detection of MpM. APTES functionalized NiCo2O4 was coated on carbon cloth and used as the working electrode. Surface functionalization with APTES on NiCo2O4 was aimed at augmenting the adsorption/interaction of MpM due to its binding properties. The developed sensor showed a very low detection limit of 1.23 nM with linear ranges of 10-100 nM and 1-100 µM and its practical applicability was examined using artificial samples of blood serum and cerebrospinal fluid, validating its potential application in real-life scenarios.

Combating Alcohol Adduct Impurity in Immunosuppressant Drug Product Manufacturing: A Scientific Investigation for Enhanced Process Control.

OBJECTIVE: This research aims to elucidate critical impurities in process validation batches of tacrolimus injection formulations, focusing on identification and characterization of previously unreported impurity at RRT 0.42, identified as the tacrolimus alcohol adduct. The potential root causes for the formation of new impurity was determined using structured risk assessment by cause and effect fishbone diagram. The primary objective was to propose mitigation plan and demonstrate the control of impurities with 6 month accelerated stability results in development batches. METHODS: The investigation utilizes method validation and characterization studies to affirm the accuracy of quantifying the tacrolimus alcohol adduct. The research methodology employed different characterization techniques like rotational rheometer, ICP‒MS, MALDI-MS, 1H NMR, 13C NMR, and DEPT-135 NMR for structural elucidation. Additionally, the exact mass of the impurity is validated using electrospray ionization mass spectra. RESULTS: Results indicate successful identification and characterization of the tacrolimus alcohol adduct. The study further explores the transformation of Tacrolimus monohydrate under various conditions, unveiling the formation of Tacrolimus hydroxy acid and proposing the existence of a novel degradation product, the Tacrolimus alcohol adduct. Six-month data from development lots utilizing Manufacturing Process II demonstrate significantly lower levels of alcohol adducts. CONCLUSIONS: Manufacturing Process II, selectively locates Tacrolimus within the micellar core of HCO-60, this prevent direct contact of ethanol with Tacrolimus which minimizes impurity alcohol adduct formation. This research contributes to the understanding of tacrolimus formulations, offering ways to safeguard product integrity and stability during manufacturing and storage.

Liquid chromatography-tandem mass spectrometry method for mycophenolic acid and its glucuronide determination in saliva samples from children with nephrotic syndrome.

BACKGROUND: Saliva sampling is one of the methods of therapeutic drug monitoring for mycophenolic acid (MPA) and its metabolite, mycophenolic acid glucuronide (MPAG). The study describes the liquid chromatography tandem mass spectrometry (LC-MS/MS) method developed for saliva MPA and MPAG determination in children with nephrotic syndrome. METHODS: The mobile phase consisted of methanol and water at gradient flow, both with 0.1% formic acid. Firstly, 100 µL of saliva was evaporated at 45 °C for 2 h to dryness, secondly, it was reconstituted in the mobile phase, and finally 10 µL was injected into the LC-MS/MS system. Saliva from ten children with nephrotic syndrome treated with mycophenolate mofetil was collected with Salivette®. RESULTS: For MPA and MPAG, within the 2-500 ng/mL range, the method was selective, specific, accurate and precise within-run and between-run. No carry-over and matrix effects were observed. Stability tests showed that MPA and MPAG were stable in saliva samples if stored for 2 h at room temperature, 18 h at 4 °C, and at least 5 months at - 80 °C as well as after three freeze-thaw cycles, in a dry extract for 16 h at 4 °C, and for 8 h at 15 °C in the autosampler. The analytes were not adsorbed onto Salivette® cotton swabs. For concentrations above 500 ng/mL, the samples may be diluted twofold. In children, saliva MPA and MPAG were within the ranges of 4.6-531.8 ng/mL and 10.7-183.7 ng/mL, respectively. CONCLUSIONS: The evaluated LC-MS/MS method has met the validation requirements for saliva MPA and MPAG determination in children with nephrotic syndrome. Further studies are needed to explore plasma-saliva correlations and assess their potential contribution to MPA monitoring.

Effect of Extraction Procedure Substitution on Automated Tacrolimus, Sirolimus, and Cyclosporine Assays.

BACKGROUND: An erroneously high tacrolimus level was reported to a clinician. A root cause analysis investigation failed to determine the cause of the error. It was suspected that the incorrect preanalytical extraction reagent and procedure was used during testing; however, how this would affect the assayed drug concentration was unclear. Here we investigated the effect of the substitution of sirolimus, tacrolimus, and cyclosporine extraction reagents on assayed drug concentration. METHODS: Tacrolimus, sirolimus, and cyclosporine concentration were measured on the Abbott Architect i2000 analyzer. Each assay requires a preanalytical extraction step, with a distinct reagent. We investigated the effect of the substitution of the extraction reagents and procedure between the 3 assays on the measured drug concentration. Two experiments were performed, one on samples of known drug concentration and one on samples with no drug present. RESULTS: Substituting cyclosporine and sirolimus extraction procedures increased assayed tacrolimus concentrations from 5.6 to 8.47 (+51.25%) and 8.13 (+45.18%) ng/mL, respectively. Extraction procedure substitutions decreased assayed sirolimus from 13.63 to 4.60 (-66.25%) and 8.07 (-40.79%) ng/mL for cyclosporine and tacrolimus. Cyclosporine concentration increased from 274.60 to 391.30 (+42.50%) ng/mL using sirolimus extraction reagents and to 757.30 (+175.78%) ng/mL using tacrolimus extraction reagents. Cross-reactivity was observed between the tacrolimus assay and sirolimus and cyclosporine extraction reagents. CONCLUSIONS: Significant changes, both positive and negative, are observed in assayed drug concentration when incorrect extraction procedures are used in the Abbott i2000 tacrolimus, sirolimus, and cyclosporine assays. Preanalytic extraction procedures should be investigated when performing root cause analysis for erroneous therapeutic drug values.

The Pharmacokinetics in Mice and Cell Uptake of Thymus Immunosuppressive Pentapeptide Using LC-MS/MS Analysis.

Thymus immunosuppressive pentapeptide (TIPP) is a novel anti-inflammatory peptide with high efficacy and low toxicity. This study aims to establish a selective LC-MS/MS method for analyzing the analyte TIPP in biological samples, laying the foundation for further PK and PD studies of TIPP. Protein precipitation was conducted in acetonitrile supplemented with 2% formic acid and 25 mg/mL dithiothreitol as a stabilizer, which was followed by backwashing the organic phase using dichloromethane. The chromatographic separation of TIPP was achieved on a C18 column with a gradient elution method. During positive electrospray ionization, TIPP was analyzed via multiple-reaction monitoring. The linear relationships between the concentration of TIPP and peak area in murine plasma cell lysates, supernatants, and the final cell rinse PBS were established within the ranges of 20−5000 ng/mL, 1−200 ng/mL, 10−200 µg/mL, and 0.1−20 ng/mL, respectively (r2 > 0.99). Validated according to U.S. FDA guidelines, the proposed method was proved to be acceptable. Such a method had been successfully applied to investigate the pharmacokinetics of TIPP in mice via subcutaneous injection. The plasma half-life in mice was 5.987 ± 1.824 min, suggesting that TIPP is swiftly eliminated in vivo. The amount of TIPP uptake by RBL-2H3 cells was determined using this method, which was also visually verified by confocal. Furthermore, the effective intracellular concentration of TIPP was deduced by comparing the intracellular concentration of TIPP and degrees of inflammation, enlightening further investigation on the intracellular target and mechanism of TIPP.

Diagnostic Value of Procalcitonin in Transplant Patients Receiving Immunosuppressant Drugs: A Retrospective Electronic Medical Record-Based Analysis.

OBJECTIVES: To evaluate concentrations of procalcitonin (PCT) in transplant recipients receiving immunosuppressive therapy compared with nonimmunosuppressed patients. METHODS: We analyzed a data set of 9,500 inpatient encounters to compare levels of PCT and other biomarkers of infection (C-reactive protein [CRP], WBC count, and absolute neutrophil count [ANC]) between immunosuppressed and nonimmunosuppressed cohorts. We also assessed the correlation between PCT and clinical variables in immunosuppressed patients. RESULTS: Patients receiving immunosuppressive drugs had significantly higher levels of maximal and minimal PCT compared with the nonimmunosuppressed patients (P < .0001 and P = .0019, respectively). However, CRP levels, WBC count, and ANC were significantly lower in immunosuppressed patients compared with the nonimmunosuppressed patients (P = .0003, P < .0019, and P = .0001, respectively). CONCLUSIONS: Our results from real-world data demonstrated that PCT dynamics remain intact despite immunosuppressive therapy, in contrast to other biomarkers such as CRP, WBC, and ANC. In addition, higher PCT levels are associated with systemic infections and reflect disease severity.

Tacrolimus concentrations after renal transplantation in a mother-neonate dyad: Maternal, neonatal and breast milk measurements.

WHAT IS KNOWN AND OBJECTIVE: We aim to add to the few reports on tacrolimus concentrations in breast milk and in maternal, umbilical vein and neonatal blood after maternal renal transplantation. CASE SUMMARY: In a 30-year-old pregnant woman, the tacrolimus concentration at delivery was the same in maternal, umbilical vein and neonatal blood. The breast milk/maternal blood tacrolimus ratio ranged from 0.40 to 0.64. WHAT IS NEW AND CONCLUSION: The maternal and neonatal blood tacrolimus concentrations at birth are equivalent; thus, one must assume that maternal tacrolimus concentrations directly affect the foetus and/or neonate. Tacrolimus is not detectable in the neonate 3 weeks after birth, suggesting that there is minimal transfer through breast milk.

Development and Validation of a Bioanalytical LC-MS/MS Method for Simultaneous Determination of Sirolimus in Porcine Whole Blood and Lung Tissue and Pharmacokinetic Application with Coronary Stents.

Sirolimus is a hydrophobic macrolide compound that has been used for long-term immunosuppressive therapy, prevention of restenosis, and treatment of lymphangioleiomyomatosis. In this study, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the simultaneous determination of sirolimus in both porcine whole blood and lung tissue. Blood and lung tissue homogenates were deproteinized with acetonitrile and injected into the LC-MS/MS system for analysis using the positive electrospray ionization mode. The drug was separated on a C18 reversed phase column with a gradient mobile phase (ammonium formate buffer (5 mM) with 0.1% formic acid and acetonitrile) at 0.2 mL/min. The selected reaction monitoring transitions of m/z 931.5 → 864.4 and m/z 809.5 → 756.5 were applied for sirolimus and ascomycin (the internal standard, IS), respectively. The method was selective and linear over a concentration range of 0.5-50 ng/mL. The method was validated for sensitivity, accuracy, precision, extraction recovery, matrix effect, and stability in porcine whole blood and lung tissue homogenates, and all values were within acceptable ranges. The method was applied to a pharmacokinetic study to quantitate sirolimus levels in porcine blood and its distribution in lung tissue following the application of stents in the porcine coronary arteries. It enabled the quantification of sirolimus concentration until 2 and 14 days in blood and in lung tissue, respectively. This method would be appropriate for both routine porcine pharmacokinetic and bio-distribution studies of sirolimus formulations.

Low Content of Cyclosporine A and Its Metabolites in the Colostrum of Post-Transplant Mothers.

The rate of post-transplant mothers who breastfeed while on immunosuppression is progressively increasing. Data on breastfeeding while on cyclosporine-based regimens are limited. Therefore, we assessed the amount of cyclosporine and its metabolites that might be ingested by a breastfed infant by measuring the concentration of cyclosporine and its metabolites in the colostrum of seven post-transplant mothers. The mean concentration of cyclosporine in the colostrum was 22.40 ± 9.43 mcg/L, and the estimated mean daily dose of the drug was 1049.22 ± 397.41 ng/kg/24 h. Only three metabolites (AM1, DHCsA, and THCsA) had mean colostrum amounts comparable to or higher than cyclosporine itself, with the daily doses being 468.51 ± 80.37, 2757.79 ± 1926.11, and 1044.76 ± 948.56 ng/kg/24 h, respectively. Our results indicate a low transfer of cyclosporine and its metabolites into the colostrum in the first two days postpartum and confirm the emerging change to the policy on breastfeeding among post-transplant mothers. A full assessment of the safety of immunosuppressant exposure via breastmilk will require further studies with long-term follow-ups of breastfed children.

Development and validation of a combined enzymatic-digestion/mass spectrometry assay for Tacrolimus quantitation in cardiac biopsies.

Recent studies report strategies for analysing immunosuppressive drugs in brain, liver and renal tissue, mostly in animals: we developed and validated a two steps combined enzymatic digestion/mass spectrometry assay to quantify Tacrolimus (TAC) in heart biopsies. Our aims were to avoid sample loss and sample contamination during the laboratory preparation, and to limit matrix effects in the electrospray ionization source (ESI) of the mass spectrometer. Enzymatic tissue digestion followed by a liquid-liquid drug extraction in the same vial of reaction allowed us to reach both our aims. The assay was assessed for selectivity, matrix effect, linearity, Lower Limit of Quantification (LLOQ) and Detection (LOD), accuracy and precision, according to the "Guideline on Bioanalytical Method Validation (EMA). A stable isotopically labelled (SIL) analogue (13CD2-TAC) was used as internal standard. The chromatographic separation of the analyte took 6 min. The observed linear range of quantification was 0.0162-0.520 ng in terms of TAC added to the biopsies (by 50 µL of the corresponding working solutions). The limit of detection and the lower limit of quantification (LLOQ) were 0.008 and 0.0162 ng, respectively. Both the mobile phases contained ammonium acetate and formic acid that promote the formation of ammoniated precursor ions that can be easily fragmented ([M + NH4]+, TAC m/z 821.3; 13CD2-TAC m/z 824.3). The calibration curves were generated by plotting analyte-to-internal standard peak area ratios versus TAC amount (ng) added to the biopsies, and using a weighted (1/x) linear regression. Curves were not forced to pass through the origin. Swine hearts were employed as blank matrix for all the analytical method validation procedures but, after approval by the ethics committee (by "Fondazione IRCCS Policlinico San Matteo": Protocol 20190032933), TAC was also quantified in endomyocardial biopsies from informed and consenting heart transplant patients. The study was funded by Fondazione IRCCS Policlinico San Matteo (RC08017617), as a part of the clinical studies on the maintenance of immunosuppressive therapy in cardiac transplant patients. Tacrolimus concentrations in patients biopsies were expressed as ratio between the detected amount of TAC (ng) in the tissue and the weight of the tissue itself (mg).

Lack of concordance between EMIT assay and LC-MS/MS for Therapeutic Drug Monitoring of Mycophenolic Acid: Potential increased risk for graft rejection?

Therapeutic drug monitoring (TDM) of immunosuppressive drugs is crucial in organ-transplanted patients to prevent rejection or toxic effects due to inadequate dosage. Mycophenolic acid (MPA) is a commonly used immunosuppressant in this setting. Nowadays, MPA concentrations are monitored by Enzyme Multiplied Immunoassay Technology (EMIT), and Liquid Chromatography (LC)-based techniques, particularly coupled to Tandem Mass Spectrometry (LC-MS/MS). This study evaluates the concordance between TDM results for MPA obtained through CE-IVD EMIT and LC-MS/MS assays in plasma samples. LC-MS/MS quantification was based on a commercial kit and the analytical performance in terms of accuracy was tested through external proficiency tests and inter-laboratory comparison with a home-made HPLC-UV method. Both these evaluations confirmed the reliability of the LC-MS/MS method (1.6 % and 9.0 % of bias, respectively). Conversely, the comparison between EMIT and LC-MS/MS showed overestimation by EMIT of 33.5 %. This bias resulted concentration-dependent, ranging from 46.4 % in the concentration range of 1-2 mg/L, to 21.4 % over 4 mg/L. Considering the theoretical clinical impact of this overestimation, a fraction comprised between 12.4 % and 31.4 % of samples which resulted over three different minimum effective concentration values by EMIT (no indication for dose adjustment) had discordant indications by LC-MS/MS (dose adjustment needed). Concluding, this study highlights a clinically relevant systematic overestimation of MPA concentration by EMIT, supporting the switch to LC-MS/MS techniques for TDM purpose. However, further prospective studies are needed in order to evaluate the clinical impact of switching the TDM activity from EMIT to LC-MS/MS in a larger cohort in a long period.

Consolidation of vitamin A and E methods onto a multiplexing liquid chromatography tandem mass spectrometry platform simplifies laboratory workflow.

BACKGROUND: Vitamin A and E are routinely monitored to assess nutritional status. The most commonly used approach for their measurement involves laborious liquid-liquid extraction followed by high-performance liquid chromatography (HPLC) analysis on dedicated instrumentation. We describe a simple, rapid protocol for measurement of vitamin A and E and their integration into an existing online sample preparation liquid chromatography tandem mass spectrometry (SPLC-MS/MS) workflow. METHODS: We performed a method comparison between the SPLC-MS/MS and HPLC methods for vitamin A and E by measuring patient specimens across the concentration range 11-81 µg/dL for vitamin A and 1-18 mg/L for vitamin E. The analysis times on each platform were also compared. RESULTS: SPLC-MS/MS and HPLC methods were comparable with regards to analytical performance; mean bias across the measured range was 2.54% (95% CL: -11.56-16.64%) for vitamin A and -2.04% (95% CL: -18.20-14.12%) for vitamin E. Total analysis times were 7 min and 15 min for SPLC-MS/MS and HPLC respectively. CONCLUSIONS: The development of a simplified sample preparation protocol and the use of multiplexing SPLC-MS/MS have reduced sample analysis times for vitamin A and E. This method has also optimized clinical workflow through consolidation of previously independent benches.

Application of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization for enhanced peptide mapping analysis of non-biological complex drug glatiramer acetate using liquid chromatography/electrospray ionization collision-induced dissociation high-resolution mass spectrometry.

RATIONALE: Glatiramer acetate (GA) (Copaxone®) is a non-biological complex drug (NBCD) comprising random-sequence polymer chains of four amino acids (MW ~ 5-9 kDa) with unknown structure. The characterization of NBCDs by reversed-phase liquid chromatography/mass spectrometry (RPLC/MS) peptide mapping is often impeded by insufficient separation and/or low sensitivity. To overcome this issue, pre-column derivatization of GA peptide digest was used to improve RPLC/MS detectability and to generate a comprehensive peptide profile. METHODS: Amino groups of peptides generated by trypsin digestion of GA were derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reagent. The derivatized mixture of random-sequence peptides was analyzed by liquid chromatography/positive-mode electrospray ionization collision-induced dissociation high-resolution mass spectrometry (RPLC/ESI-CID-HRMS/MS). Data-independent LC/MSE mode was used for data acquisition. RESULTS: The derivatization of the GA peptide mixture increased the detectability of RPLC/ESI-CID-HRMS/MS analysis. The efficacy of the procedure was demonstrated by using selected peptides related to different polymeric chain origins. The resultant peptides were derivatized in a predictable manner giving a minimum of side products. The reproducibility of the developed method was demonstrated by comparing peptide elution profiles derived from six Copaxone® lots. CONCLUSIONS: Application of the AQC pre-column derivatization provides a framework that could be used as an attractive approach for monitoring the quality and characterization of NBCD products in the pharmaceutical industry.

Synthesis of bifunctional cabbage flower-like Ho3+/NiO nanostructures as a modifier for simultaneous determination of methotrexate and carbamazepine.

Cabbage flower-like Ho3+/NiO nanostructure (CFL-Ho3+/NiO NSs) with significant electrocatalytic oxidation has been published for the first time. First, structure and morphology of CFL-Ho3+/NiO-NSs have been described by XRD, SEM, and EDX methods. Then, CFL-Ho3+/NiO-NSs have been applied as a modifier for simultaneous electrochemical detection of methotrexate (MTX) and carbamazepine (CBZ). Functions of the modified electrode have been dealt with through electrochemical impedance spectroscopy (EIS). It has been demonstrated that the electrode response has been linear from 0.001-310.0 µM with a limit of detection of 5.2 nM and 4.5 nM (3 s/m) through DPV for MTX and CBZ. Diffusion coefficient (D) and heterogeneous rate constant (kh) have been detected for MTX and CBZ oxidation at the surface of the modified electrode. Moreover, CFL-Ho3+/NiO-NS/GCE has been employed for determining MTX and CBZ in urine and drug specimens. Outputs showed the analyte acceptable recovery. Therefore, the electrode could be applied to analyze both analytes in drug prescription and clinical laboratories. Graphical abstract Electrochemical sensor based on bifunctional cabbage flower-like Ho3+/NiO nanostructures modified glassy carbon electrode for simultaneous detecting methotrexate and carbamazepine was fabricated.

Facile fabrication of a novel disposable pencil graphite electrode for simultaneous determination of promising immunosuppressant drugs mycophenolate mofetil and tacrolimus in human biological fluids.

An innovative electrochemical sensor was proposed for simultaneous determination of mycophenolate mofetil (Mph) and tacrolimus (TAC) for the first time. A novel sensor based on electro-polymerization of multi-walled carbon nanotubes (MWCNTs) and a novel Cu-1N-allyl-2-(2,5-dimethoxyphenyl)-4,5-diphenyl-1H-imidazole metal organic framework (Cu-ADPPI MOF) on disposable pencil graphite electrode (dPGE). Many techniques were used to characterize the electrochemical activity and surface structure of the fabricated sensor. The proposed sensor exhibited good catalytic performance towards Mph and TAC oxidation due to the synergistic effect. Under optimal conditions, the proposed sensor has achieved a linear range of 0.85-155 × 10-8 M and 1.1-170.0 × 10-8 M with LODs of 0.28 × 10-8 M and 0.36 × 10-8 M for Mph and TAC, respectively. The designated sensor showed good reproducibility, repeatability, stability, and selectivity for the determination of Mph and TAC. Moreover, the simultaneous determination of Mph and TAC in different human biological fluids was carried out with acceptable results. As a result, the proposed sensor opens a new venue for the use of electro-polymerized MOFs in combination with other conductive materials such as MWCNTs for electrochemical sensing of different analytes with the desired sensitivity and selectivity. Graphical abstract Construction of disposable graphite electrode, based on electro-deposition of multilayer films of multi-walled carbon nanotubes and a new generation of Cu-MOFs, for simultaneous analysis of tacrolimus and mycophenolate mofetil for the first time.

A 6 Second Analytical Method for Quantitation of Tacrolimus in Whole Blood by Use of Laser Diode Thermal Desorption Tandem Mass Spectrometry.

BACKGROUND: Therapeutic drug monitoring of immunosuppressive drugs is imperative for organ transplant recipients. High-performance LC-MS/MS is considered gold standard; however, immunoassays provide rapid turnaround time. New technology was developed to reduce mass spectrometry analytical run-time. The laser diode thermal desorption source coupled with tandem mass spectrometry (LDTD-MS/MS) eliminates chromatographic separation to increase analytical throughput. METHODS: A rapid, 6 second, LDTD-MS/MS analytical method was developed for the quantification tacrolimus in whole blood. Whole blood samples were lysed, followed by protein precipitation and solid-phase extraction. Extracted samples with desorption solution were spotted onto a LazWell plate then dried and loaded into the LDTD source for analysis with an AB SCIEX 5500 mass spectrometer in positive multiple reaction monitoring mode. The LDTD laser profile ramps from 0% to 65% of full power over 3 s and is held at 65% for 1 s before returning to initial conditions for 2 s. RESULTS: Data presented include tacrolimus by LDTD-MS/MS comparison to LC-MS/MS, sensitivity, imprecision, interference, linearity, and stability. Method comparison between LDTD-MS/MS and a validated in-house LC-MS/MS assay yielded the following: (LDTD-MS/MS) = 1.119 (LC-MS/MS) + 0.23 ng/mL, Sy/x = 1.26, r = 0.9871 (n = 122). The limit of quantification by LDTD-MS/MS for tacrolimus was <0.3 ng/mL and total imprecision was <10%. CONCLUSIONS: Laser diode thermal desorption tandem mass spectrometry technology can provide rapid turnaround time to result for tacrolimus. The analytical time for LDTD-MS/MS was 6 s compared to 135 s by LC-MS/MS, a >95% decrease in analytical time.

A multidrug LC-MS/MS method for the determination of five immunosuppressants in oral fluid.

Aim: This study aimed: to develop and validate an LC-MS/MS method for mycophenolic acid, tacrolimus, sirolimus, everolimus and cyclosporin A in oral fluid (OF), as an essential tool to study the usefulness of OF as an alternative matrix for immunossuppressants' therapeutic drug monitoring; and to find the best OF collector for these analytes. Materials & Methods: Chromatographic separation was achieved using an XBridge® Shield RP18 analytical column maintained at 65ºC, using 2 mM ammonium formate and 0.1% formic acid in water (A) and acetonitrile (B) as mobile phase. OF sample was extracted with solid phase extraction after sonication and protein precipitation. Results & Conclusions: Method validation met all the acceptance criteria. LODs were 0.05-1 ng/ml, and LOQs 0.1-5 ng/ml. Silanized tubes offered the best recoveries. The method was successfully applied to 31 OF specimens, describing everolimus detection in OF for the first time. Conclusion: The proposed method is sensitive enough for the detection of OF trough concentrations in patients receiving immunosuppressants when using an appropriate OF collector.