Surrogate Analyte Approach Research Articles

Development and validation of a HILIC-MS/MS method for the quantitation of fructose in human urine in support of clinical programs

In a previous publication [1], a 20-minute UPLC®-MS/MS method, employing a surrogate analyte approach, was developed and validated to measure fructose and sorbitol, as mechanistic biomarkers, in human plasma to support first-in-human (FIH) studies. Different from plasma which maintains its homeostasis, urine has no such homeostasis mechanisms [2], therefore it is expected to be able to accommodate more changes. Here we describe the development and validation of a LC-MS/MS method for the quantiation of fructose in human urine to support clinical trials. A hydrophilic interaction chromatography (HILIC) method using an Asahipak NH2P-50 column (Shodex, 4.6 × 250 mm, 5 µm) was developed. Acetone precipitation was utilized to extract fructose from urine. For validation, stable isotope-labeled 13C6-fructose was used as the surrogate analyte for fructose in the preparation of calibration curves. QCs were prepared using both the surrogate analyte (13C6-fructose) and the authentic analyte (fructose). Difficulties were encountered for post-extraction stability experiments especially for authentic fructose QCs at low concentrations. Extensive troubleshooting revealed that fructose’s chromatography improved as the column aged. As a result, the response factor of fructose increased over time for low concentration samples, leading to failed post-extraction stability experiments. A column cleaning procedure was implemented to ensure consistency in chromatography performance. The HILIC-MS/MS method was successfully validated and applied to analyze clinical samples with a 91% overall run passing rate.

A validated surrogate analyte LC-MS/MS assay for quantification of endogenous cortisol in human whole blood

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 13C3-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 13C3-cortisol, and accuracy, precision and robustness were all acceptable per current regulatory guidance for bioanalytical method validation of chromatographic assays for cortisol- and 13C3-cortisol-based quality control (QC) samples when quantified against a 13C3-cortisol calibration curve. The acceptable robustness of cortisol-based QCs when quantified against a 13C3-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.

Quantitation of 2-hydroxyglutarate in human plasma via LC-MS/MS using a surrogate analyte approach.

Aim: 2-Hydroxyglutarate (2-HG) is a target engagement biomarker in patients after treatment with inhibitors of mutated isocitrate dehydrogenase (mIDH). Accurate measurement of 2-HG is critical for monitoring the inhibition effectiveness of the inhibitors. Materials & methods: Human plasma samples were spiked with stable isotope labelled internal standard, processed by protein precipitation, and analyzed using LC-MS/MS. This method was validated following regulatory guidance and has been successfully applied in a clinical study for mIDH inhibition. Results: An LC-MS/MS method with a surrogate analyte approach was developed and validated to measure 2-HG in human plasma with acceptable intra- and inter-assay accuracy and precision. Conclusion: A sensitive and robust LC-MS/MS method was developed and validated for measuring 2-HG in human plasma.

A novel dried blood spots analysis combined with on-spot reaction for determination of trimethylamine N-oxide and its related compounds

The application of dried blood spots in clinical research is becoming increasingly popular owing to its convenient collection, storage, and transportation compared to that of conventional biological samples. The potential of trimethylamine N-oxide and its related compounds as biomarkers for various cardiovascular diseases, such as atherosclerosis, stroke, thrombosis, and heart failure, was recently highlighted, which was the driving force behind the development of an analytical method to identify trimethylamine N-oxide and eight related compounds in dried blood spots. In the proposed method, a novel “on-spot reaction” approach was introduced to overcome the low loading efficiency of trimethylamine in dried blood spots. Upon the addition of 50 μL of blood onto the filter paper pretreated with dilute HCl, an acid–base neutralization reaction in the blood spots transformed the volatile trimethylamine to a salt. Next, a punched disc with a diameter of 6.0 mm was eluted by agitation with 20 mM ammonium formate for 10 min and derivatized with 1.0 M ethyl bromoacetate at 80 °C for 60 min. A surrogate analyte approach was employed for quantification of these endogenous compounds in the complex matrix. Analysis was carried out using zwitterionic hydrophilic interaction liquid chromatography–high-resolution mass spectrometry. The established method was validated and applied to monitor real samples from 30 clinical cases. The proposed new methodology based on dried blood spots could greatly improve the convenience, analytical sensitivity, and selectivity of cardiovascular disease testing.

A double surrogate approach for the quantitation of 2-Hydroxyglutarate – An oncometabolite in human brain tumors via LC-MS/MS

We are reporting a validated LC–MS/MS assay for the quantitation of 2-Hydroxyglutarate (2-HG), an endogenous oncometabolite, in human brain tumors. To address the challenges of endogenous levels of 2-HG in biological matrix and the limited availability of human brain tumors, a surrogate analyte in conjunction with a surrogate matrix approach was used (stable isotope labeled 2-HG in monkey brain homogenate). Human brain tumor homogenate samples were spiked with stable isotope labeled internal standard, processed by protein precipitation, and analyzed using LC–MS/MS. The mass spectrometer was optimized to ensure equal molar response between 2-HG – the authentic analyte and 13C5 2-HG – the surrogate analyte. Parallelism was successfully demonstrated between human brain tumor homogenate (the authentic matrix) and monkey brain homogenate (the surrogate matrix). The linear analytical range of the assay was set at 0.15–150 μM. This validated LC–MS/MS method demonstrated excellent accuracy and precision. For 13C5 2-HG in monkey brain homogenate and human brain tumor homogenate, the overall accuracy was between 98.4 % and 102 %, and the intra- and inter-assay precision (%CV) were less than 5.7 %. 2-HG was found to be stable in human brain tumor homogenate for at least 26 h at room temperature, 454 days when stored at 70 °C and after five freeze/thaw cycles. The assay has been successfully applied to human brain tumor samples to support clinical studies.Novel Aspect: A surrogate matrix and surrogate analyte approach to quantify endogenous 2-HG concentrations in human brain tumors.

Surrogate analyte-based quantification of main endocannabinoids in whole blood using liquid chromatography-tandem mass spectrometry.

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.

Quantification of free fatty acids in human stratum corneum using tandem mass spectrometry and surrogate analyte approach.

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.

Development of an Analytical Method for Dibutyl Phthalate Determination Using Surrogate Analyte Approach.

Dibutyl phthalate (DBP) is a phthalic acid ester and is widely used in polymeric products to make them more flexible. DBP is found in almost every plastic material and is believed to be persistent in the environment. Various analytical methods have been used to measure DBP in different matrices. Considering the ubiquitous nature of DBP, the most important challenge in DBP analyses is the contamination of even analytical grade organic solvents with this compound and lack of availability of a true blank matrix to construct the calibration line. Standard addition method or using artificial matrices reduce the precision and accuracy of the results. In this study a surrogate analyte approach that is based on using deuterium labeled analyte (DBP-d4) to construct the calibration line was applied to determine DBP in hexane samples.

Surrogate analyte approach for quantitation of endogenous NAD(+) in human acidified blood samples using liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

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 0.5N perchloric acid at a ratio of 1:3 (v:v, blood:perchloric acid) during sample collection. 25μL 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)C5-NAD(+) was used as the surrogate analyte for authentic analyte, NAD(+). The standard curve ranging from 0.250 to 25.0μg/mL in acidified human blood for (13)C5-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)C5-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)C5-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.

A surrogate analyte-based LC–MS/MS method for the determination of γ-hydroxybutyrate (GHB) in human urine and variation of endogenous urinary concentrations of GHB

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 (0.1–1μg/ml). The limit of detection and the limit of quantification of 2H6-GHB were 0.05 and 0.1μg/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 4h 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 1.8μg/ml and from 4.5 to 530μg/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.

Quantification of free and total desmosine and isodesmosine in human urine by liquid chromatography tandem mass spectrometry: A comparison of the surrogate-analyte and the surrogate-matrix approach for quantitation

In spite of the data suggesting the potential of urinary desmosine (DES) and isodesmosine (IDS) as biomarkers for elevated lung elastic fiber turnover, further validation in large-scale studies of COPD populations, as well as the analysis of longitudinal samples is required. Validated analytical methods that allow the accurate and precise quantification of DES and IDS in human urine are mandatory in order to properly evaluate the outcome of such clinical studies. In this work, we present the development and full validation of two methods that allow DES and IDS measurement in human urine, one for the free and one for the total (free+peptide-bound) forms. To this end we compared the two principle approaches that are used for the absolute quantification of endogenous compounds in biological samples, analysis against calibrators containing authentic analyte in surrogate matrix or containing surrogate analyte in authentic matrix. The validated methods were employed for the analysis of a small set of samples including healthy never-smokers, healthy current-smokers and COPD patients. This is the first time that the analysis of urinary free DES, free IDS, total DES, and total IDS has been fully validated and that the surrogate analyte approach has been evaluated for their quantification in biological samples. Results indicate that the presented methods have the necessary quality and level of validation to assess the potential of urinary DES and IDS levels as biomarkers for the progression of COPD and the effect of therapeutic interventions.

Surrogate Matrix And Surrogate Analyte Approaches For Definitive Quantitation of Endogenous Biomolecules

Quantitation of biomarkers by LC-MS/MS is complicated by the presence of endogenous analytes. This challenge is most commonly overcome by calibration using an authentic standard spiked into a surrogate matrix devoid of the target analyte. A second approach involves use of a stable-isotope-labeled standard as a surrogate analyte to allow calibration in the actual biological matrix. For both methods, parallelism between calibration standards and the target analyte in biological matrix must be demonstrated in order to ensure accurate quantitation. In this communication, the surrogate matrix and surrogate analyte approaches are compared for the analysis of five amino acids in human plasma: alanine, valine, methionine, leucine and isoleucine. In addition, methodology based on standard addition is introduced, which enables a robust examination of parallelism in both surrogate analyte and surrogate matrix methods prior to formal validation. Results from additional assays are presented to introduce the standard-addition methodology and to highlight the strengths and weaknesses of each approach. For the analysis of amino acids in human plasma, comparable precision and accuracy were obtained by the surrogate matrix and surrogate analyte methods. Both assays were well within tolerances prescribed by regulatory guidance for validation of xenobiotic assays. When stable-isotope-labeled standards are readily available, the surrogate analyte approach allows for facile method development. By comparison, the surrogate matrix method requires greater up-front method development; however, this deficit is offset by the long-term advantage of simplified sample analysis.

Quantification of endogenous steroids in human urine by gas chromatography mass spectrometry using a surrogate analyte approach

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.

Assessment of endogenous androgen levels in meat, liver and testis of Iranian native cross-breed male sheep and bull by gas chromatography-mass spectrometry

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.

Accurate quantification of endogenous androgenic steroids in cattle's meat by gas chromatography mass spectrometry using a surrogate analyte approach

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.