Determining of 18 amino acids in plasma of pregnant women with sleep disorders by UHPLC-MS/MS.

Many pregnant women experience sleep disorders, and amino acid levels could play a crucial role in affecting maternal sleep. To explore this potential relationship, an accurate and effective UHPLC-MS/MS method has been developed to monitor 18 amino acids in the plasma samples of pregnant women. This method aims to assess how plasma amino acid levels might be linked to sleep disorders during pregnancy. Plasma samples were precipitated with acetonitrile containing 0.2% formic acid. We used 5% seralbumin as the surrogate matrix to establish quantitative curves for amino acid determination in human plasma. The method was validated in both the surrogate matrix and human plasma. The optimized UHPLC-MS/MS method was validated, showing that that the analytes had comparable recovery and negligible matrix effects in both 5% seralbumin and human plasma. The linearity, lower limit of quantification, precision, accuracy, and stability all met the acceptance criteria. The validated method was successfully applied to determination of the plasma levels of 18 amino acids in pregnant women with or without sleep disorders, indicating that alanine, lysine, tryptophan, glutamic acid, and phenylalanine levels had significant changes which may be related to sleep disorders during early pregnancy. An accurate, reliable, and efficient UHPLC-MS/MS method was successfully developed and support to find the specific amino acids as potential biomarkers for sleep disorders in pregnant women.

Single Isotopologue for In-Sample Calibration and Absolute Quantitation by LC-MS/MS.

Targeted mass spectrometry (MS)-based absolute quantitative analysis has been increasingly used in biomarker discovery. The ability to accurately measure the masses by MS enabled the use of isotope-incorporated surrogates having virtually identical physiochemical properties with the target analytes as calibrators. Such a unique capacity allowed for accurate in-sample calibration. Current in-sample calibration uses multiple isotopologues or structural analogues for both the surrogate and the internal standard. Here, we simplified this common practice by using endogenous light peptides as the internal standards and used a mathematical deduction of "heavy matching light, HML" to directly quantify an endogenous analyte. This method provides all necessary assay performance parameters in the authentic matrix, including the lower limit of quantitation (LLOQ) and intercept of the calibration curve, by using only a single isotopologue of the analyte. This method can be applied to the quantitation of proteins, peptides, and small molecules. Using this method, we quantified the efficiency of heart tissue digestion and recovery using sodium deoxycholate as a detergent and two spiked exogenous proteins as mimics of heart proteins. The results demonstrated the robustness of the assay.

Quantification of glucocorticoid and progestogen metabolites in bovine plasma, skimmed milk and saliva by UHPLC-HR-MS with polarity switching.

Steroid metabolites are increasingly in focus when searching for novel biomarkers in physiological mechanisms and their disorders. While major steroids such as progesterone and cortisol are well-researched and routinely determined to assess the health, particularly the reproductive status of mammals, the function of potentially biologically active progestogen and glucocorticoid metabolites is widely unexplored. One of the main reasons for this is the lack of comprehensive, sensitive, and specific analytical methods. This is particularly the case when analyzing matrices like milk or saliva obtained by non-invasive sampling with steroid concentrations often below those present in plasma. Therefore, a new UHPLC-HR-MS method based on an Ultimate UHPLC system equipped with an Acquity HSS T3 reversed-phase column and a Q Exactive™ mass spectrometer was developed, enabling the simultaneous chromatographic separation, detection and quantification of eleven isobaric glucocorticoids (11-dehydrocorticosterone (A), corticosterone (B), cortisol (F), cortisone (E), the tetrahydrocortisols (THF): 3α,5α-THF, 3α,5ß-THF, 3ß,5α-THF, 3ß,5ß-THF, and the tetrahydrocortisones (THE): 3α,5α-THE, 3α,5ß-THE, 3ß,5α-THE) and twelve progestogens (progesterone (P4), pregnenolone (P5), the dihydroprogesterones (DHP): 20α-DHP, 20ß-DHP, 3α-DHP, 3ß-DHP, 5α-DHP, 5ß-DHP, and the tetrahydroprogesterones (THP): 3α,5α-THP, 3α,5ß-THP, 3ß,5α-THP, 3ß,5ß-THP) in bovine plasma, skimmed milk, and saliva. A simple liquid-liquid extraction (LLE) with MTBE (methyl tert-butyl ether) was used for sample preparation of 500 µL plasma, skimmed milk, and saliva. Heated electrospray ionization (HESI) with polarity switching was applied to analyze steroids in high-resolution full scan mode (HR-FS). The method validation covered the investigation of sensitivity, selectivity, curve fitting, carry-over, accuracy, precision, recovery, matrix effects and applicability. A high sensitivity in the range of pg mL-1 was achieved for all steroids suitable for the analysis of authentic samples.

Expanding assay range to accommodate a monoclonal antibody therapeutic quantification in blood and cerebrospinal fluid.

Antibody therapeutic levels in neurodegenerative diseases are often measured in both serum and cerebrospinal fluid (CSF). Due to 0.1% drug partition from serum to CSF and the higher sensitivity needs, usually two different assays are required. The different Gyrolab Bioaffy compact discs can extend the dynamic range of assays. Here, an assay was developed and adapted on two different Gyrolab Bioaffy compact discs (200 and 4000 nl) to achieve the required sensitivity and assay dynamic range needed for the measurement of drug in both serum and CSF. This was accomplished by using the same critical reagents with minimal assay development to transition from a serum to a CSF assay.

Lessons learned from regulatory submissions involving endogenous therapeutic analyte bioanalysis.

Endogenous therapeutic analytes include hormones, neurotransmitters, vitamins, fatty acids and inorganic elements that are naturally present in the body because either the body produces them or they are present in the normal diet. The accurate measurement of endogenous therapeutic analytes poses a challenge when the administered exogenous therapeutic analyte and its endogenous counterpart cannot be distinguished. In this article, real case examples with endogenous therapeutic analyte bioanalysis during drug development in support of regulatory submissions are collected and presented. The article highlights common challenges encountered and lessons learned related to bioanalysis of endogenous therapeutic analytes and provides practical tips and strategies to consider from a regulatory perspective.

A universal surrogate matrix assay for urea measurement in clinical pharmacokinetic studies of respiratory diseases.

In pharmacokinetic studies for respiratory diseases, urea is a commonly used dilution marker for volume normalization of various biological matrices, owing to the fact that urea diffuses freely throughout the body and is minimally affected by disease states. In this study, we developed a convenient liquid chromatography-tandem mass spectrometry (LC-MS/MS) surrogate matrix assay for accurate urea quantitation in plasma, serum and epithelial lining fluid. Different mass spectrometer platforms and ionization modes were compared in parallel. The LC method and mass spectrometer parameters were comprehensively optimized to reduce interferences, to smooth the baseline and to maximize the signal-to-noise ratio. Saline was selected as the surrogate matrix, and its suitability was confirmed by good parallelism and accurate quality control sample measurements. Reliable and robust assay performance was demonstrated by precision and accuracy, dilution integrity, sensitivity, recovery and stability, all of which met bioanalysis requirements to support clinical studies. The assay performance was also verified and better understood by comparing it with a colorimetric assay and to a surrogate analyte assay. The newly developed surrogate matrix assay has the potential to be further expanded for urea quantitation in numerous physiological matrices.

Simultaneous determination of UDCA and its major metabolites in human plasma with surrogate matrix by a rapid and specific LC-MS/MS method.

A rapid, convenient, and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of ursodeoxycholic acid (UDCA), and its major metabolites, glycoursodeoxycholic acid (GUDCA) and tauroursodeoxycholic acid (TUDCA) in human plasma. Methanol was chosen as surrogate matrix for preparation the calibrators to establish calibration curves. Isotope internal standard was used for each analyte. After plasma samples were deproteinized with methanol, the post-treatment samples were analyzed on a ZORBAX SB-C18 column (2.1 × 50 mm, 1.8 µm) with 2 mM ammonium acetate and acetonitrile as mobile phase at a flow rate of 0.5 mL/min. Detection was performed on a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) employing negative ESI interface using API5500 triple quadrupole tandem mass spectrometer system, with the transitions set at m/z 391.4 â†’ m/z 391.4, m/z 448.3 â†’ m/z 73.9, m/z 498.4 â†’ m/z 80.1, m/z 395.3 â†’ m/z 395.3, m/z 453.3 â†’ m/z 74.0, and m/z 503.2 â†’ m/z 79.9 for UDCA, GUDCA, TUDCA, UDCA-d4, GUDCA-d5, and TUDCA-d5, respectively. The calibration curve ranges were 5.00-2500 ng/mL for UDCA and GUDCA and 0.500-250 ng/mL for TUDCA. The intra- and inter-day precision was within 7.00% in terms of relative standard deviation (RSD%) and the accuracy within 11.75% in terms of relative error. The selectivity, sensitivity, extraction recovery, matrix effect, dilution reliability, and stability were within the acceptable range. The method was successfully applied to a pharmacokinetic study in 12 healthy Chinese volunteers after oral administration of 250 mg UDCA.

From fundamentals in calibration to modern methodologies: A tutorial for small molecules quantification in liquid chromatography-mass spectrometry bioanalysis.

Over the last two decades, liquid chromatography coupled to mass-spectrometry (LC‒MS) has become the gold standard to perform qualitative and quantitative analyses of small molecules. When quantitative analysis is developed, an analyst usually refers to international guidelines for analytical method validation. In this context, the design of calibration curves plays a key role in providing accurate results. During recent years and along with instrumental advances, strategies to build calibration curves have dramatically evolved, introducing innovative approaches to improve quantitative precision and throughput. For example, when a labeled standard is available to be spiked directly into the study sample, the concentration of the unlabeled analog can be easily determined using the isotopic pattern deconvolution or the internal calibration approach, eliminating the need for multipoint calibration curves. This tutorial aims to synthetize the advances in LC‒MS quantitative analysis for small molecules in complex matrices, going from fundamental aspects in calibration to modern methodologies and applications. Different work schemes for calibration depending on the sample characteristics (analyte and matrix nature) are distinguished and discussed. Finally, this tutorial outlines the importance of having international guidelines for analytical method validation that agree with the advances in calibration strategies and analytical instrumentation.

Advancement in Analytical Strategies for Quantification of Biomarkers with a Special Emphasis on Surrogate Approaches.

Accurate quantification of biomarkers has always been a challenge for many bioanalytical scientists due to their endogenous nature and low concentration in biological matrices. Different analytical approaches have been developed for quantifying biomarkers including enzyme-linked immunosorbent assay, immunohistochemistry, western blotting, and chromatographic techniques assisted with mass spectrometry. Liquid chromatography-tandem mass spectrometry-based quantification of biomarkers has gained more attention over other traditional techniques due to its higher sensitivity and selectivity. However, the primary challenge lies with this technique includes the unavailability of a blank matrix for method development. To overcome this challenge, different analytical approaches are being developed including surrogate analyte and surrogate matrix approach. Such approaches include quantification of biomarkers in a surrogate matrix or quantification of an isotopically labeled surrogate analyte in an authentic matrix. To demonstrate the authenticity of the surrogate approach, it is mandatory to establish quantitative parallelism through validation employing respective surrogate analytes and surrogate matrices. In this review, different bioanalytical approaches for biomarker quantification and recent advancements in the field aiming for improvement in the specificity of the techniques have been discussed. Liquid chromatography-tandem mass spectrometry-based surrogate approaches for biomarker quantification and significance of parallelism establishment in both surrogate matrix and surrogate analyte-based approaches have been critically discussed. In addition, different methods for demonstrating parallelism in the surrogate method have been explained.

UHPLC-ESI-MS/MS assay for quantification of endocannabinoids in cerebrospinal fluid using surrogate calibrant and surrogate matrix approaches.

Endocannabinoids are endogenous lipids with the main function recognized to act as neuromodulators through their cannabinoid receptors. Dysregulation of the endocannabinoid system is implicated in various pathologies, such as inflammatory and neurodegenerative diseases. In this study we describe a sensitive UHPLC-MS/MS method for the analysis of trace levels of 7 endocannabinoids in cerebrospinal fluid samples. The analytes covered comprised 1- and 2-arachidonoylglycerol 1- and 2-AG (which were analysed as sum due to their interconversion), 2-arachidonylglycerol ether 2-AGE, anandamide AEA, N-linoleoyl ethanolamide LEA, N-palmitoyl ethanolamide PEA and N-oleoyl ethanolamide OEA. Analytes were extracted from the biofluid by a simple monophasic procedure involving protein precipitation with acetonitrile (MeCN). The analytical method is based on chromatographic separation of the analytes with solid-core (core-shell, superficially porous) particle column Cortecs C18+ . Gradient elution with changing proportion of water and acetonitrile and constant concentration of formic acid provided reasonable separation of analytes, close elution of analytes and their internal standards and minimized matrix effects in biological samples. For specific detection of the endocannabinoids a triple-quadrupole tandem mass spectrometer with electrospray ionisation (ESI) and selected reaction monitoring (SRM) mode was used, and it provided good assay selectivity. The developed method required a minute volume of the biological samples (50 µL) and achieved excellent sensitivity (the lower limit of detection was between 4.15 and 30.18 pM of the biological sample). Linear calibration was achieved in the range from 25 to 10,545 pM for AEA, 90-3802 pM for 1-AG, 90-724 pM for 2-AG, 12-5226 pM for LEA, 33-13,942 for OEA, 34-23,850 pM for 2-AGE, 72-30,190 for PEA and 10-4218 for AEA-d4 in CSF. The method was validated and revealed relative errors in the range of - 14.7 to + 12.3% at LLOQ and - 14.1 to + 14.2% for the remaining validation range. Precisions were in the acceptable range (< 20% RSD at LLOQ, and <15% for the remaining levels) as well. It was finally used to quantify endocannabinoids in human cerebrospinal fluid obtained from 118 donors. Accurate quantification of endogenous compounds in biological samples was achieved by using two different principal approaches (surrogate matrix for AEA, 2-AG, OEA, 2-AGE, LEA and PEA, and surrogate calibrant for AEA only) and they were evaluated by use of the Passing-Bablok regression. Concentrations (median) of CSF samples of patients suffering from CNS infection and controls were found to be around 160 pM for 1- and 2-AG, 86 pM for AEA, 62 for 2-AGE, 58 for LEA, 93 pM for PEA, and 83 pM for OEA.

Bioanalysis of INCB000928 in hemodialysate: prevention of nonspecific binding and validation of surrogate matrices.

Aim: To develop and validate a bioanalytical method for the quantification of INCB000928 in hemodialysate. Materials & methods: Blank dialysate and phosphate-buffered saline were compared with hemodialysate for surrogate matrix selection. Direct addition of internal standard without analyte extraction and a high-performance LC-MS/MS were used for analysis. Results & conclusion: INCB000928 in hemodialysate exhibited strong nonspecific binding to polypropylene containers. In the presence of 10% isopropyl alcohol, the loss of INCB000928 was fully recovered, regardless of pre- or post-addition of the solvent. Blank dialysate and phosphate-buffered saline were determined to be appropriate surrogate matrices by using a three-way cross-comparison and were subsequently validated in the quantitative analysis of INCB000928 in hemodialysate.

Fibrodysplasia ossificans progressiva (FOP) is a very rare disease characterized by congenital malformation of the great toes and progressive heterotopic ossification. The genetic cause of FOP is mutation in the gene ALK2. INCB000928 is a novel and orally available drug that inhibits ALK2 protein activity and has been shown to prevent ossification in a laboratory mouse model of FOP. Patients with end-stage renal disease who undergo hemodialysis may require a different dose of INCB000928. This study showed that INCB000928 was heavily adsorbed by the container wall, resulting in underestimated drug levels in hemodialysate. We present a method to accurately measure INCB000928 levels in hemodialysate by using isopropyl alcohol as an antiadsorption agent and cost-effective surrogate matrix.

Quantitation of endogenous GnRH by validated nano-HPLC-HRMS method: a pilot study on ewe plasma.

Gonadotropin-releasing hormone isoform I (GnRH), a neuro-deca-peptide, plays a fundamental role in development and maintenance of the reproductive system in vertebrates. The anomalous release of GnRH is observed in reproductive disorder such as hypogonadotropic hypogonadism, polycystic ovary syndrome (PCOS), or following prenatal exposure to elevated androgen levels. Quantitation of GnRH plasma levels could help to diagnose and better understand these pathologies. Here, a validated nano-high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) method to quantify GnRH in ewe plasma samples is presented. Protein precipitation and solid-phase extraction (SPE) pre-treatment steps were required to purify and enrich GnRH and internal standard (lamprey-luteinizing hormone-releasing hormone-III, l-LHRH-III). For the validation process, a surrogate matrix approach was chosen following the International Council for Harmonisation (ICH) and FDA guidelines. Before the validation study, the validation model using the surrogate matrix was compared with those using a real matrix such as human plasma. All the tested parameters were analogous confirming the use of the surrogate matrix as a standard calibration medium. From the validation study, limit of detection (LOD) and limit of quantitation (LOQ) values of 0.008 and 0.024 ng/mL were obtained, respectively. Selectivity, accuracy, precision, recovery, and matrix effect were assessed with quality control samples in human plasma and all values were acceptable. Sixteen samples belonging to healthy and prenatal androgen (PNA) exposed ewes were collected and analyzed, and the GnRH levels ranged between 0.05 and 3.26 ng/mL. The nano-HPLC-HRMS developed here was successful in measuring GnRH, representing therefore a suitable technique to quantify GnRH in ewe plasma and to detect it in other matrices and species.

Criteria of slope and endogenous level for selection of matrix in the quantitation of endogenous compounds by LC-MS/MS.

Selection of the appropriate matrix for standard curve is critical for an accurate and sensitive biomarker method. Slope of a standard curve is the key factor for parallelism assessment between tested matrix and authentic matrix for LC-MS/MS assays. Here the authors have established slope criteria using a generic equation and endogenous level criteria for achieving assay sensitivity. The slope difference criterion is from -13.0 to +17.6% for LC-MS assays with ± 15% bias criteria. When the ratio of endogenous concentration in the tested matrix to the lower limit of quantitation is <4.0, the lower limit of quantitation is achievable. If these criteria are met, the tested matrix can be used for assays. The utility of the two criteria has been illustrated with case studies.

Therapeutic drug monitoring of caffeine and its primary metabolites in plasma using LC-ESI-MS/MS for apnea of prematurity treatment: Evaluation of ultrapure water as a surrogate matrix.

The growing evidence has endorsed the view that therapeutic drug monitoring of caffeine for apnea of prematurity is helpful for dose tailoring when the therapeutic response is lacking or toxicity is suspected. However, plasma without caffeine is difficult to obtain. Therefore, a method was developed and validated to measure caffeine and its three primary metabolites (paraxanthine, theobromine and theophylline) using LC-ESI-MS/MS in human plasma and several surrogate matrices. The chromatographic separation of analytes was finally achieved on a Waters Symmetry C18 (4.6 × 75 mm, 3.5 µm) column. Several strategies were successfully applied to overcome the matrix effects: (a) appropriate dilution for sample cleanup; (b) a starting lower proportion of organic phase; and (c) multiple individual stable-labeled isotopic internal standards. The parallelism between the authentic matrix and surrogate matrices was convincing. The recovery of the analytes in both human plasma and rat plasma was acceptable over the linear range (0.500-50.0 µg/ml for caffeine and 0.0100-1.00 µg/ml for three metabolites). The method was successfully applied in 118 samples from 74 preterm infants with apnea of prematurity. The rat plasma or ultrapure water as a surrogate matrix is worthy of recommendation for routine therapeutic drug monitoring of caffeine.

An Improved Method to Quantify Short-Chain Fatty Acids in Biological Samples Using Gas Chromatography-Mass Spectrometry.

Gut microbial metabolites, short-chain fatty acids (SCFAs), are found at multiple locations in the host body and are identified as important metabolites in gut microbiome-associated diseases. Quantifying SCFAs in diverse biological samples is important to understand their roles in host health. This study developed an accurate SCFA quantification method by performing gas chromatography-mass spectrometry (GC/MS) in human plasma, serum, feces, and mouse cecum tissue. The samples were acidified with hydrochloric acid, and the SCFAs were extracted using methyl tert-butyl ether. In this method, distilled water was selected as a surrogate matrix for the quantification of SCFAs in target biological samples. The method was validated in terms of linearity, parallelism, precision, recovery, and matrix effect. The developed method was further applied in target biological samples. In conclusion, this optimized method can be used as a simultaneous SCFA quantification method in diverse biological samples.

A validated LC-MS/MS method for the quantitation of cefazolin in human adipose tissue: Application of EMR-Lipid sorbent as an efficient sample clean-up before mass spectrometric analyses.

A novel, simple, rapid, and sensitive high-performance liquid chromatography-tandem mass spectrometry method was developed to determine cefazolin concentrations in human adipose tissue. Sample preparation was performed by protein precipitation followed by using Captiva EMR-Lipid plates. The mobile phase consisted of 5 mM ammonium formate and 0.1% formic acid in water and 0.1% formic acid in ACN, and was pumped through a Synergi Fusion-RP column with a gradient elution program at a flow rate of 0.3 mL/min. The mass spectrometer was operated in a positive ion mode. Cloxacillin was used as an internal standard due to the observed cross-signal contribution between cefazolin and 13C2,15N-cefazolin. The method was validated according to the FDA and EMA guidelines and passed all the acceptance criteria. The calibration range was 0.05-50 µg/mL in adipose tissue homogenate (0.15-150 µg/g in adipose tissue), precision CV < 4.5%, accuracy within 93.1-100.4%. The carry-over was negligible, recovery of the method was high, and no significant matrix effect was present. Rat subcutaneous adipose tissue was demonstrated to be a suitable surrogate matrix for human adipose tissue. The validated method was successfully applied in a pilot pharmacokinetic study and will further be used in a large cohort of non-obese and obese patients dosed prophylactically with cefazolin before surgeries.

A developed HPLC-MS/MS method to quantitate 5 steriod hormones in clinical human serum by using PBS as the surrogate matrix.

Steroid hormones play an essential role in regulating physiological and reproductive development throughout the lifetime of an individual. One of the difficulties in determining endogenous substances is the lack of a blank matrix. Especially when the level of analytes is lower than the level in the so-called blank matrix. In the present study, an optimized HPLC-MS/MS method was developed and validated to quantify androstenedione (ASD), testosterone (Ts), dehydroepiandrosterone (DHEA), 5α-dihydrotestosterone (DHT), and progesterone (P) in serum samples from healthy people using PBS (pH = 7.4) as the blank surrogate matrix. Simultaneously, the method investigated the characteristics of NaCl, bull serum albumin, pure water as surrogate matrices for the analysis of steroid hormones. The data showed that the matrix effects of ASD, Ts, DHEA, DHT, and P in the same groups were not significantly different between PBS and twice charcoal-stripped serum (CS2S) as a blank surrogate matrix. Furthermore, the LLOQ using PBS as the blank matrix was up to 0.005 ng/mL for ASD, Ts, and P and 0.05 ng/mL for DHEA and DHT. The reference ranges of concentration (CPBS) of 5 steroid hormones were provided. Compared to the concentration with CS2S (CCSS) as the blank surrogate matrix, the relative biases (RBs) of Ts, DHT, P, and DHEA were finally stabilized at approximately -0.7%, -15%, -1.2%, and 9.2%, respectively. The results suggest that, in the cases of special required, the developed HPLC-MS/MS method can be used to determine the absolute concentration of 5 hormones in biological samples with PBS as the blank surrogate matrix.

Evaluation of multiple hydrophilic interaction chromatography columns and surrogate matrix for arginine quantification in saliva by high-resolution mass spectrometry.

Arginine, a pivotal ingredient in many biochemical synthetic pathways, can be used as a biomarker for many oral care clinical applications. It is still a challenge to develop a sensitive and reliable chromatographic method to quantify arginine as a biomarker in saliva, with or without arginine product pretreatment. The current method solved two critical issues for arginine quantitation in human saliva. The first issue was how to optimize arginine peak shape. A hydrophilic interaction chromatography method based on the column selection, pH and pKa relationship, mobile phase ionic strength, organic solvent consideration, and temperature effects was developed. An optimized chromatographic condition for arginine quantitation in the saliva matrix was obtained. The second issue was how to build confidence in the use of a simple surrogate matrix methodology to replace the more complex traditional standard addition methodology. The surrogate matrix methodology we developed is applicable to the measurement of arginine as a potential non-invasive biomarker in human saliva. The method detection and quantification limit reached 2 and 6 ng/mL. The tailing factor was within the 0.9-1.1 range even though arginine had three pKa values at 2.18, 9.09, and 13.2.

Simultaneous Quantification of 3'- and 6'-Sialyllactose in Rat Plasma Using Liquid Chromatography-Tandem Mass Spectrometry and Its Application to a Pharmacokinetic Study.

Sialyllactose (SL), an acidic oligosaccharide, has immune-protective effects against pathogens and helps with the development of the immune system and intestinal microorganisms. To elucidate the pharmacokinetic characterization after oral administration to rats, the simultaneous quantification method for 3'-SL and 6'-SL in rat plasma was validated, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an electrospray ionization (ESI) mode. Several types of columns [C18, amide, and hydrophilic interaction liquid chromatography (HILIC) phase] were used to separate the peaks of 3'-SL and 6'-SL, which improved chromatographic selectivity. Ultimately, the HILIC phase column had a good peak shape and quick resolution, with a mobile phase comprising ammonium acetate buffer and acetonitrile obtained by gradient elution. In addition, the simultaneous quantification of 3'-SL and 6'-SL in rat plasma samples were adequately applied to pharmacokinetic study.

A Surrogate Matrix-Based Approach Toward Multiplexed Quantitation of an sGC Stimulator and cGMP in Ocular Tissue and Plasma.

A liquid chromatography-tandem mass spectrometry assay was developed and qualified for the multiplexed quantitation of a small molecule stimulator of soluble guanylate cyclase (sGC) and its target engagement biomarker, 3',5'-cyclic guanosine monophosphate (cGMP), in ocular tissues and plasma from a single surrogate matrix calibration curve. A surrogate matrix approach was used in this assay due to the limited quantities of blank ocular matrices in a discovery research setting. After optimization, the assay showed high accuracy, precision, and recovery as well as parallelism between the surrogate matrix and the biological matrices (rabbit plasma, vitreous, and retina-choroid). This assay provided pharmacokinetic and target engagement data after intravitreal administration of the sGC stimulator. The nitric oxide-sGC-cGMP pathway is a potential target to address glaucoma. Increasing sGC-mediated production of cGMP could improve aqueous humor outflow and ocular blood flow. The sGC stimulator showed dose-dependent exposure in rabbit vitreous, retina-choroid, and plasma. The cGMP exhibited a delayed yet sustained increase in vitreous humor but not retina-choroid. Multiplexed measurement of both pharmacokinetic and target engagement analytes reduced animal usage and provided improved context for interpreting PK and PD relationships.