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Stable isotope‐labeled polyene phosphatidylcholines (PCs) were required for quantitative bioanalytical assays of products extracted from soybeans. A facile and efficient method for the synthesis of both [D9] and [D13]2,3‐dilinoleoyl‐sn‐glycero‐3‐phosphocholine ([D9] & [D13]DLPC) in excellent yields and high isotope‐enrichment has been developed applying cheap and commercially available glycine, 2‐aminoethanol and iodomethane‐d3 (99 %, 2H) as starting materials and deuterium sources. [D9]DLPC and [D13]DLPC can be used as an internal standard and/or as a surrogate analyte. [ABSTRACT FROM AUTHOR]

Bear bile is a valuable medicinal material used in traditional Chinese medicine for over 2000 years. However, developing a substitute has become necessary because of protection measures for this endangered species. The ingredients of in vitro cultured bear bile powder (CBBP) include tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA, and it has pharmacological properties that are similar to those of natural bear bile powder (NBBP). In this study, the pharmacokinetic parameters of both CBBP and NBBP were measured in rats with a new surrogate analyte LC–MS method using stable isotopes as surrogate analytes (D4‐TUDCA, D4‐TCDCA, D4‐UDCA and D4‐CDCA) with response factors validated in authentic matrix (plasma) for simultaneously monitoring the authentic analytes (TUDCA, TCDCA, UDCA and CDCA). The method validation was satisfactory for the linear regression (r, 0.9975–0.9994), precision (RSD intra‐day, 0.72–9.35%; inter‐day, 3.82–9.02%), accuracy (RE, −12.42–5.67%) and matrix effect (95.53–99.80%), along with analyte recovery (95.90–98.82%) and stability (89.48–101.81%) of surrogate analytes, and precision (RSD intra‐day, 1.06– 11.51%; inter‐day, 2.23– 11.38%), accuracy (RE, −7.40–10.76%) and stability (87.37–111.70%) of authentic analytes. We successfully applied this method to evaluate the pharmacokinetics of CBBP and NBBP in rats, which revealed the critical in vivo properties of both bear bile preparations. [ABSTRACT FROM AUTHOR]

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.

Online SPE LC-MS with 13C3-cortisol as a surrogate analyte was developed, validated and applied for obtaining hair cortisol levels of 114 healthy volunteers. The first 3 cm from a posterior vertex position were analyzed as three individual segments resulting in 310 hair cortisol levels, which were used, after log transformation, to predict a range of values observed in future. The median value of hair cortisol was 4.76 pg/mg. The employed method allowed simple processing, high throughput and may be readily expanded to analyze additional steroid compounds in hair. [ABSTRACT FROM AUTHOR]

Abstract: As biomarkers, endogenous neurotransmitters play critical roles in the process of neuropsychiatric diseases, and neurotransmitter levels in different brain regions can contribute to neurological disease diagnosis and treatment. Due to the lack of a blank matrix for endogenous neurotransmitters, surrogate‐matrix and surrogate‐analyte approaches have been used for the determination of neurotransmitters to solve this problem. In this study, we capitalised on the high accuracy, precision, and throughput of UHPLC‐MS/MS and developed new methods based on the two approaches. Both approaches satisfied FDA and EMA validation criterias after an appropriate parallelism assessment, and they were used to further quantify the three endogenous neurotransmitters, including dopamine (DA), serotonin (5‐HT) and γ‐aminobutyric acid (GABA) in rat brain four regions (cortex, striatum, hypothalamus and hippocampus) which represent the catecholamines, indolamines, and amino acids, respectively. Comparison of the results in the same rats (n = 10) showed there was no significant difference in DA, 5‐HT, or GABA levels between the two approaches (P > 0.05). The concentrations of DA and GABA were highest in striatum and hypothalamus, respectively, and the levels of 5‐HT were paralleled in striatum and hippocampus almost 2‐fold higher than other regions. This is the first study to compare these two approaches in the determination of endogenous neurotransmitter content in the rat brain, and the surrogate‐matrix approach proved to be simple, rapid, and reliable, considering cost, matrix similarity, and practicality. [ABSTRACT FROM AUTHOR]

The early diagnosis of diabetic nephropathy (DN) is rather challenging. Our previous study suggested that citric acid is a potential marker for the early diagnosis of diabetic nephropathy in db/db mice. For the first time, in this study, a surrogate analyte of 13 C 6 -citric acid was employed to generate calibration curves for the quantitative measurement of the endogenous citric acid in the sera of db/db mice and diabetic nephropathy patients by GC/MS after the analytes were extracted, methoximated and trimethylsilylated. The constant response factor of 13 C 6 -citric acid versus citric acid over the linear range indicated the identical ionization efficiency of these two compounds. The full validation assessments suggested that the method is sensitive, specific, reliable, reproducible and has acceptable parameters. Statistical analysis revealed cut-off citric acid concentrations of 29.24 μg/mL with a 95% confidence interval between 32.75 and 39.16 μg/mL in the diabetic nephropathy patients and 16.74 and 22.57 μg/mL in the normal controls. The areas under the receiver operating characteristic curves indicated accuracies of over 90% for the diagnoses of early diabetic nephropathy in both humans and db/db mice, which suggests that the serum citric acid level is potentially a biomarker that could assist in the diagnosis of diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Abstract: The free fatty acids (FFAs) are one of the major components of the lipids in the stratum corneum (SC), the uppermost layer of the skin. Relative composition of FFAs has been proposed as a biomarker of the skin barrier status in patients with atopic dermatitis (AD). Here, we developed an LC‐ESI‐MS/MS method for simultaneous quantification of a range of FFAs with long and very long chain length in the SC collected by adhesive tape (D‐Squame). The method, based on derivatization with 2‐bromo‐1‐methylpyridinium iodide and 3‐carbinol‐1‐methylpyridinium iodide, allowed highly sensitive detection and quantification of FFAs using multiple reaction monitoring. For the quantification, we applied a surrogate analyte approach and internal standardization using isotope labeled derivatives of FFAs. Adhesive tapes showed the presence of several FFAs, which are also present in the SC, a problem encountered in previous studies. Therefore, the levels of FFAs in the SC were corrected using C12:0, which was present on the adhesive tape, but not detected in the SC. The method was applied to SC samples from patients with atopic dermatitis and healthy subjects. Quantification using multiple reaction monitoring allowed sufficient sensitivity to analyze FFAs of chain lengths C16–C28 in the SC collected on only one tape strip. [ABSTRACT FROM AUTHOR]

A robust high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) assay was developed and qualified for the measurement of cyclic nucleotides (cNTs) in rat brain tissue. Stable isotopically labeled 3′,5′-cyclic adenosine- 13 C 5 monophosphate ( 13 C 5 -cAMP) and 3′,5′-cyclic guanosine- 13 C, 15 N 2 monophosphate ( 13 C 15 N 2 -cGMP) were used as surrogate analytes to measure endogenous 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP). Pre-weighed frozen rat brain samples were rapidly homogenized in 0.4 M perchloric acid at a ratio of 1:4 (w/v). Following internal standard addition and dilution, the resulting extracts were analyzed using negative ion mode electrospray ionization LC–MS/MS. The calibration curves for both analytes ranged from 5 to 2000 ng/g and showed excellent linearity (r 2 > 0.996). Relative surrogate analyte-to-analyte LC–MS/MS responses were determined to correct concentrations derived from the surrogate curves. The intra-run precision (CV%) for 13 C 5 -cAMP and 13 C 15 N 2 -cGMP was below 6.6% and 7.4%, respectively, while the inter-run precision (CV%) was 8.5% and 5.8%, respectively. The intra-run accuracy (Dev%) for 13 C 5 -cAMP and 13 C 15 N 2 -cGMP was <11.9% and 10.3%, respectively, and the inter-run Dev% was <6.8% and 5.5%, respectively. Qualification experiments demonstrated high analyte recoveries, minimal matrix effects and low autosampler carryover. Acceptable frozen storage, freeze/thaw, benchtop, processed sample and autosampler stability were shown in brain sample homogenates as well as post-processed samples. The method was found to be suitable for the analysis of rat brain tissue cAMP and cGMP levels in preclinical biomarker development studies. [ABSTRACT FROM AUTHOR]

Acetycholinesterase (AChE) that regulates hydrolysis of acetylcholine (ACh) in the brain, is an important target for treatment of Alzheimer's disease (AD), a feature of which is ACh deficiency. However, the methods to precisely determine AChE activity are still under development. We developed a new method to exploit acetylcholine-d4 as a surrogate substrate of ACh and measure product choline-d4 via liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay detected activity of AChE present in the normal mouse brain, which is consistent with the standard Ellman assay that determines products spectrophotometrically. In AD mouse models, the result of LC-MS/MS assay showed significant higher AChE activity than that seen in control normal mice, while treatment of AD mice with an AChE inhibitor, huperzine A, led to partial decreases in AChE activity. Our results suggest that this surrogate-based LC-MS/MS method is a new, sensitive and convenient assay for the determination of AChE activity, providing a useful means for screening active compounds that target AChE. [ABSTRACT FROM AUTHOR]

Accurate quantitative analysis of endogenous analytes is essential for several clinical and non-clinical applications. LC–MS/MS is the technique of choice for quantitative analyses. Absolute quantification by LC/MS requires preparing standard curves in the same matrix as the study samples so that the matrix effect and the extraction efficiency for analytes are the same in both the standard and study samples. However, by definition, analyte-free biological matrices do not exist for endogenous compounds. To address the lack of blank matrices for the quantification of endogenous compounds by LC–MS/MS, four approaches are used including the standard addition, the background subtraction, the surrogate matrix, and the surrogate analyte methods. This review article presents an overview these approaches, cite and summarize their applications, and compare their advantages and disadvantages. In addition, we discuss in details, validation requirements and compatibility with FDA guidelines to ensure method reliability in quantifying endogenous compounds. The standard addition, background subtraction, and the surrogate analyte approaches allow the use of the same matrix for the calibration curve as the one to be analyzed in the test samples. However, in the surrogate matrix approach, various matrices such as artificial, stripped, and neat matrices are used as surrogate matrices for the actual matrix of study samples. For the surrogate analyte approach, it is required to demonstrate similarity in matrix effect and recovery between surrogate and authentic endogenous analytes. Similarly, for the surrogate matrix approach, it is required to demonstrate similar matrix effect and extraction recovery in both the surrogate and original matrices. All these methods represent indirect approaches to quantify endogenous compounds and regardless of what approach is followed, it has to be shown that none of the validation criteria have been compromised due to the indirect analyses. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • A detailed tutorial of the fundamentals of LC‒MS quantification is provided. • A collection of over 150 references on calibration is given. • Relative, semi and absolute quantification are described. • Methodological differences among external and internal calibrations are highlighted. • Applications with regard to method validation guidelines are discussed. [ABSTRACT FROM AUTHOR]

A high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) assay for the quantitative determination of NAD + in human whole blood using a surrogate analyte approach was developed and validated. Human whole blood was acidified using 0.5 N perchloric acid at a ratio of 1:3 (v:v, blood:perchloric acid) during sample collection. 25 μL of acidified blood was extracted using a protein precipitation method and the resulting extracts were analyzed using reverse-phase chromatography and positive electrospray ionization mass spectrometry. 13 C 5 -NAD + was used as the surrogate analyte for authentic analyte, NAD + . The standard curve ranging from 0.250 to 25.0 μg/mL in acidified human blood for 13 C 5 -NAD + was fitted to a 1/ x 2 weighted linear regression model. The LC–MS/MS response between surrogate analyte and authentic analyte at the same concentration was obtained before and after the batch run. This response factor was not applied when determining the NAD + concentration from the 13 C 5 -NAD + standard curve since the percent difference was less than 5%. The precision and accuracy of the LC–MS/MS assay based on the five analytical QC levels were well within the acceptance criteria from both FDA and EMA guidance for bioanalytical method validation. Average extraction recovery of 13 C 5 -NAD + was 94.6% across the curve range. Matrix factor was 0.99 for both high and low QC indicating minimal ion suppression or enhancement. The validated assay was used to measure the baseline level of NAD + in 29 male and 21 female human subjects. This assay was also used to study the circadian effect of endogenous level of NAD + in 10 human subjects. [ABSTRACT FROM AUTHOR]

A bioanalytical strategy for the simple and accurate determination of endogenous substances in a variety of biological matrices using liquid chromatography-tandem mass spectrometry is described. The robust method described here uses two stable isotope-labeled compounds as a surrogate analyte and an internal standard to construct calibration curves with authentic matrices that can be applied to determine N -acetyl- l -aspartyl- l -glutamic acid (NAAG) levels in rat brain, plasma, and cerebrospinal fluid (CSF) using a simple extraction and with a short analysis time of 4 min. The validated lower limits of quantification were 1.00 nmol/g for brain and 0.0100 nmol/mL for plasma and CSF. Using this method, regional differences in NAAG levels in the brain as well as plasma and CSF levels that were much lower than those in the brain were successfully confirmed in treatment-naïve rats. Moreover, after the rats were treated with the intraventricular administration of a NAAG peptidase inhibitor, the NAAG levels increased rapidly and dramatically in the CSF and slightly in the plasma in a time-dependent manner, while the brain levels were not affected. Thus, the procedure described here was easily applied to the determination of NAAG in different matrices in the same manner as that used for xenobiotics, and this method would also be easily applicable to the accurate measurement of endogenous substances in a variety of biological matrices. [ABSTRACT FROM AUTHOR]

The endocannabinoids (eCBs), N-arachidonoylethanolamine (anandamide, AEA) and 2-ararchidonylglycerol (2-AG) have been identified as main endogenous ligands for cannabinoid receptors. Developing a sensitive and robust method to determine AEA and 2-AG has been shown to be essential to understand their effects in stress regulation and the pathogenesis of affective disorders. Since eCBs are endogenous molecules, there is no true blank matrix available to construct calibration curves, thus, it has been a challenge to determine eCBs in plasma and brain matrix. A liquid chromatography tandem mass spectrometry (LC–MS/MS) method is developed to determine the concentrations of AEA and 2-AG in murine plasma and different brain substructures (prefrontal cortex, hippocampus and hypothalamus). To overcome the endogenous interference, a “surrogate analyte” approach was adopted using stable isotope-labeled standards as surrogates of authentic analytes to generate calibration curves in biological matrix. The mobile phase, composed of formic acid 0.1% in water–acetonitrile (40:60, v/v), was optimized to separate 2-AG and its non-bioactive isomer 1-AG. The analytes were extracted with ethyl acetate/n-hexane (9:1, v/v) and separated on an Xbridge C18 (2.1 × 100 mm, 3.5 μm) column using N-Oleoylethanolamine-d2 (OEA-d2) as the internal standard. Detection was performed in multiple reaction monitoring (MRM) mode with an electrospray ionization source operated in positive ion mode. The method was applied to assess plasma and brain eCBs in tumor-bearing mice. [ABSTRACT FROM AUTHOR]

γ-Hydroxybutyrate (GHB) is a drug of abuse with a strong anesthetic effect; however, proving its ingestion through the quantification of GHB in biological specimens is not straightforward due to the endogenous presence of GHB in human blood, urine, saliva, etc. In the present study, a surrogate analyte approach was applied to accurate quantitative determination of GHB in human urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to overcome this issue. For this, ²H6-GHB and 13C2-dl-3-hydroxybutyrate were used as a surrogate standard and as an internal standard, respectively, and parallelism between the surrogate analyte approach and standard addition was investigated at the initial step. The validation results proved the method to be selective, accurate, and precise, with acceptable linearity within calibration ranges (0.1-1 μg/ml). The limit of detection and the limit of quantification of ²H6-GHB were 0.05 and 0.1 μg/ml, respectively. No significant variations were observed among urine matrices from different sources. The stability of ²H6-GHB was satisfactory under sample storage and in-process conditions. However, in vitro production of endogenous GHB was observed when the urine sample was kept under the in-process condition for 4 h and under the storage conditions of 4 and -20 °C. In order to facilitate the practical interpretation of urinary GHB, endogenous GHB was accurately measured in urine samples from 79 healthy volunteers using the surrogate analyte-based LC-MS/MS method developed in the present study. The unadjusted and creatinine-adjusted GHB concentrations in 74 urine samples with quantitative results ranged from 0.09 to 1.8 μg/ml and from 4.5 to 530 μg/mmol creatinine, respectively. No significant correlation was observed between the unadjusted and creatinine-adjusted GHB concentrations. The urinary endogenous GHB concentrations were affected by gender and age while they were not significantly influenced by habitual smoking, alcohol drinking, or caffeine-containing beverage drinking. [ABSTRACT FROM AUTHOR]

A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed, using testosterone-d3 as a surrogate analyte, for the simultaneous quantification of goserelin and testosterone in rat plasma. According to this method, the pharmacokinetic and pharmacodynamic data were obtained from a single plasma sample aliquot. The method involved the addition of alarelin as an internal standard (IS) for goserelin and testosterone-13C3 for testosterone or testosterone-d3. The conditions for the separation of these two compounds were achieved on a ZORBAX Eclipse Plus C18 column (Agilent, 2.1×50mm, 1.8µm, Stockport, UK) in a single chromatographic run at a flow rate of 400µL/min. In order to minimize interferences of complex matrix, the extraction of plasma consisted of a protein precipitation step using methanol, followed by purification using an Oasis® HLB solid-phase extraction column. The method was validated in the concentration range of 0.01-30.0ng/mL for goserelin and 0.05-30.0ng/mL for testosterone-d3, respectively. The within- and between-run precisions were 1.7-9.2% and 2.1-6.9%, respectively. The within- and between-run accuracies were -1.8 to 5.3% and -4.9 to 4.0%, respectively. This accurate and highly specific assay provides a useful method to evaluate the pharmacokinetics and pharmacodynamics of goserelin in rats. [ABSTRACT FROM AUTHOR]

Highlights: [•] Synthesis of deuterated- and 18O-labeled DES and IDS standards. [•] Method development for free and total DES and IDS analysis in human urine. [•] Full method validation according to FDA and EMA regulations. [•] Comparison of surrogate matrix and surrogate analyte approaches. [•] Validated method application to the analysis of clinical samples. [Copyright &y& Elsevier]

ABSTRACT A simple, robust and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine the concentration of corticosterone (Cort) which is usually regarded as a stress biomarker in mouse serum. Since Cort is an endogenous hormone, a 'surrogate analyte' strategy was adopted using the stable isotope-deuterated corticosterone as a surrogate of the authentic analyte to generate the calibration curve. With telmisartan as the internal standard, the analytes were extracted with methanol, ethanol and acetone (1:1:1, v/v/v) and separated on a XTerra C18 (2.1 × 50 mm, 3.5 µm) column using a mobile phase consisting of 0.2% formic acid in water-methanol (30:70, v/v). Detection was performed in multiple reaction monitoring mode with an electrospray ionization source operated in positive ion mode. The standard curves were linear ( r2 > 0.999) over the dynamic range of 8.60-430 ng/mL, with a lower limit of quantification of 8.60 ng/mL. The intra- and inter-assay precisions were less than 15.0% of the relative standard deviation. This method was further used for analysis of serum samples from C57B/L tumor-bearing mice before and after the treatment of fluoxetine. Validation of the assay and its application to the analysis demonstrated that the method was applicable to determine meaningful changes in Cort concentrations in serum samples of the tumor-bearing mice for the stress status evaluation. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: A bioanalytical method for determining endogenous d-serine levels in the mouse brain using a surrogate analyte and liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. [2,3,3-2H]d-serine and [15N]d-serine were used as a surrogate analyte and an internal standard, respectively. The surrogate analyte was spiked into brain homogenate to yield calibration standards and quality control (QC) samples. Both endogenous and surrogate analytes were extracted using protein precipitation followed by solid phase extraction. Enantiomeric separation was achieved on a chiral crown ether column with an analysis time of only 6min without any derivatization. The column eluent was introduced into an electrospray interface of a triple–quadrupole mass spectrometer. The calibration range was 1.00 to 300nmol/g, and the method showed acceptable accuracy and precision at all QC concentration levels from a validation point of view. In addition, the brain d-serine levels of normal mice determined using this method were the same as those obtained by a standard addition method, which is time-consuming but is often used for the accurate measurement of endogenous substances. Thus, this surrogate analyte method should be applicable to the measurement of d-serine levels as a potential biomarker for monitoring certain effects of drug candidates on the central nervous system. [Copyright &y& Elsevier]

Abstract: Cholinergic dysfunction is known as a hallmark feature of Alzheimer''s disease (AD). Measurement of endogenous acetylcholine (ACh) in specific brain regions is important in understanding the pathology of AD and in designing and evaluating novel cholinomimetic agents for the treatment of AD. Since ACh is an endogenous neurotransmitter, there is no real blank matrix available to construct standard curves. It has been a challenging task to determine ACh in complex brain matrices. To overcome these difficulties, we employed a surrogate analyte strategy using ACh-d4 instead of ACh to generate calibration curves and Ch-d9 as internal standard (IS). The brain samples were deproteinized by acetonitrile with IS. Analytes and IS were separated by a HILIC column with the mobile phase composed of 20mM ammonium formate in water–acetonitrile (30:70, v/v, adjusted to pH 3.0 with formic acid) and monitored in multiple reaction monitoring (MRM) mode using a positive electrospray source. The concentrations of endogenous ACh were calculated based on the peak area ratio of the analyte to the IS using a regression equation for the corresponding surrogate standard (ACh-d4). The lower limit of detection was 0.2ng/mL and linearity was maintained over the range of 10–1000ng/mL. Compared to other currently available methods, this approach offers improved accuracy and precision for efficient analysis of ACh. The proposed method was proved successfully by evaluating the action of typical acetylcholinesterase inhibitor huperzine A in senescence accelerated mouse prone 8 (SAMP8). [Copyright &y& Elsevier]

Abstract: Providing “real blank sample” is a problem in determination of endogenous steroids in complex matrices. A new quantification strategy is proposed in the present study, which is based on using isotope-labeled steroids instead of natural steroids for constructing calibration line. This approach is called surrogate analyte and it is shown that its accuracy is better than some of the previously described methods at low concentrations and comparable to standard addition method at medium and high concentration levels. The method was fully validated to satisfy the ICH criteria and it was applied for determination of endogenous steroids in several urine samples. [Copyright &y& Elsevier]

Androgenic steroids always exist in different animal tissues at trace level, with significant numbers of interfering compounds, which makes their determination difficult. To solve some of the problems in quantification of the natural steroids in those tissues, a new GC-MS method was developed in this study. By using a surrogate analyte approach, which was developed in the authors' previous studies, and extensive sample preparation procedure, which successfully eliminates many of the interfering compounds and resulting in a cleaner extract, accuracy, precision, sensitivity and selectivity of the method for the determination of steroids in complex matrices such as meat, liver and testis were improved. By aid of this method, the levels of androgens in different tissues of Iranian native cross-breed bulls and male sheep were determined. According to the results obtained in the present study, although the androgenic profile (contents and ratios of precursors and metabolites to the main hormones) is similar between the same tissues of both animals, the total androgenic content of each tissue is higher in the bull than the same tissue in male sheep. In addition, in both animals higher amount of androgens were found in liver in comparison with meat and testis. [ABSTRACT FROM AUTHOR]

Abstract: Determination of endogenous steroids in complex matrices such as cattle''s meat is a challenging task. Since endogenous steroids always exist in animal tissues, no analyte-free matrices for constructing the standard calibration line will be available, which is crucial for accurate quantification specially at trace level. Although some methods have been proposed to solve the problem, none has offered a complete solution. To this aim, a new quantification strategy was developed in this study, which is named “surrogate analyte approach” and is based on using isotope-labeled standards instead of natural form of endogenous steroids for preparing the calibration line. In comparison with the other methods, which are currently in use for the quantitation of endogenous steroids, this approach provides improved simplicity and speed for analysis on a routine basis. The accuracy of this method is better than other methods at low concentration and comparable to the standard addition at medium and high concentrations. The method was also found to be valid according to the ICH criteria for bioanalytical methods. The developed method could be a promising approach in the field of compounds residue analysis. [Copyright &y& Elsevier]

Abstract: Chromatographic methods are increasingly being used for the quantitative determination of endogenous compounds in biological samples. This article presents an overview of the specific issues, which have to be taken into account for the development, validation and application of these methods. The usual lack of analyte-free samples of the biological matrix implies that alternative strategies for calibration have to be followed. This article compares and discusses the advantages and disadvantages of two strategies – the use of the authentic analyte in a surrogate matrix and the use of a surrogate analyte in the authentic matrix. In addition, it highlights important aspects of the validation of chromatographic methods for endogenous analytes. [Copyright &y& Elsevier]

• First known surrogate analyte-based assay for cortisol in human whole blood. • Fit-for-purpose LC-MS/MS method validated in accordance with FDA and EMA guidance. • Cortisol is stable in blood up to 25 h at RT, boding well for at-home collection. • Whole blood and plasma cortisol concentrations demonstrate promising compatibility. • First step towards supporting at-home sampling for cortisol in clinical trials. Cortisol is a steroid hormone that is frequently measured as a marker of stress, inflammation, and immune function. While commonly analyzed in saliva, hair, blood plasma and urine, a recent trend towards whole blood-based at-home collection devices has emerged, which necessitates development of more sensitive assays for cortisol in whole blood. To support the implementation of a patient-centric sampling approach in a drug development program, a fit-for-purpose surrogate analyte-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for cortisol in whole blood was developed using 13C 3 -cortisol as a surrogate analyte and cortisol-d6 as the internal standard. The surrogate analyte approach was chosen due to a lack of available cortisol-free whole blood and the absence of appropriately representative surrogate matrices. Samples were prepared using supported liquid extraction, and the LC-MS/MS analysis consisted of a 4.00 min analytical run. The method demonstrated linearity between 0.500 and 500 ng/mL of 13C 3 -cortisol, and accuracy, precision and robustness were all acceptable per current regulatory guidance for bioanalytical method validation of chromatographic assays for cortisol- and 13C 3 -cortisol-based quality control (QC) samples when quantified against a 13C 3 -cortisol calibration curve. The acceptable robustness of cortisol-based QCs when quantified against a 13C 3 -cortisol-based calibration curve also suggests parallelism between the analytes. These results indicate a viable surrogate analyte method, that is fit-for-purpose to analyze whole blood cortisol levels using a surrogate analyte LC-MS/MS approach. Evaluation of patient samples showed very promising comparability between whole blood and plasma cortisol concentrations, suggesting that whole blood could be used in place of or in addition to a plasma-based sampling protocol in clinical trials analyzing cortisol. Overall, this method presents a novel tool that is a first step in supporting the trend towards sample miniaturization and at-home sample collection, and may be readily used in clinical and diagnostic settings. [ABSTRACT FROM AUTHOR]

To meet the increasing clinical needs for 25‐hydroxyvitamin D3 (25OH‐D3) detection, the development of an efficient and accurate high‐performance liquid chromatography–mass spectrometry (HPLC–MS) method for plasma 25OH‐D3 quantitation is important. Since 25OH‐D3 is an endogenous compound, the lack of a plasma blank increases the difficulty of accurately quantifying 25OH‐D3. Selection of a method suitable for clinical monitoring among various methods for endogenous compound quantification is necessary. Methyl tert butyl ether was chosen for the sample treatment in a liquid–liquid extraction protocol. Water as a blank matrix, 5% human serum albumin in water as a blank matrix, surrogate analyte and background subtraction were designed to address the problem of a deficiency of a plasma blank. Four liquid chromatography–tandem mass spectrometry methods were fully validated to verify the advantages and limitations owing to regulatory deficiencies for endogenous compound validation. All four methods met the criteria and could be used to monitor clinical samples. Overall 30 human plasma samples were quantified in parallel using the four methods. The difference between any two methods was <12.6% and the total relative standard deviation was <5.2%. Background subtraction and 5% human serum albumin in water as a blank matrix may be better choices considering data quality, matrix similarity, cost and practicality. [ABSTRACT FROM AUTHOR]

Endocannabinoids (eCBs) are endogenous ligands of the endocannabinoid system that are known to regulate several physiological and behavioral processes. Previous studies have developed methods for the detection of main eCBs including arachidonylethanolamide (AEA) and 2‐arachidonoylglycerol (2‐AG), mostly in serum or plasma. Whole blood is a superior biomaterial for eCBs analysis owing to the nature of the shortened isolation procedure and decreased risk of 2‐AG isomerization during preparation. In this study, a surrogate analyte‐based liquid chromatography–tandem mass spectrometry assay was developed for the measurement of AEA, 2‐AG and its isomer 1‐arachidonoylglycerol (1‐AG) using a maximum of 100 μL whole blood. Chromatographic separation was achieved using a reverse‐phase column and a gradient elution. Detection was performed in selected reaction monitoring mode with an electrospray ionization source. The limits of detection of three eCBs were 0.05–0.1 ng/mL. Good linearity was observed over the concentration range. Intra‐ and inter‐assay accuracy and precision were ≤10.9 and ≤8.7% at four quality control levels. The response factor and parallelism experiment illustrated that the surrogate analytes were suitable for accurate quantification of the main eCBs in whole blood. This surrogate analyte approach was successfully applied to authentic blood samples obtained from alcohol negative drivers and those under the influence of alcohol. [ABSTRACT FROM AUTHOR]

Background: Creatinine is an endogenous compound generated from creatine by normal muscular metabolism. It is an important indicator of renal function and the serum level is routinely monitored in clinical labs. Results & methodology: Surrogate analyte (d3-creatinine) was used for calibration standard and quality control preparation and the relative instrument response ratio between creatinine and d3-creatinine was used to calculate the endogenous creatinine concentrations., Conclusion: A fit-for-purpose strategy of using a surrogate analyte and authentic matrix was adopted for this validation. The assay was the first human plasma assay using such strategy and was successfully applied to a clinical study to confirm a transient elevation of creatinine observed using an existing clinical assay.