Stable isotope dilution quantification of mutagens in cooked foods by combined liquid chromatography-thermospray mass spectrometry

A method of general applicability for the detection and quantification of mutagens in cooked foods at the ppb level is presented. A minimal sample prefractionation is employed and [Me-²H₃-labeled analogs of the compounds of interest are added for identification and quantification of mutagens by accurate measurement of chromatographic retention (K′) in reverse-phase high-performance liquid chromatography (HPLC), and by measurement of the ratio of response of the protonated molecular ions of analyte and internal standard by directly coupled liquid chromatography-mass spectrometry (LC/MS). Initial application is demonstrated in the analysis of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quonoline (MeIQ) in broiled salmon. Measured levels of IQ and MeIQ in broiled salmon flesh were 0.3–1.8 ppb and 0.6–2.8 ppb, respectively, and for the skin of broiled salmon 1.1–1.7 ppb and 1.5–3.1 ppb, respectively. Results on cooked beef and sardine are also reported.     

Identification and determination of pirlimycin residue in bovine milk and liver by high-performance liquid chromatography-thermospray mass spectrometry

Determinative and confirmatory methods of analysis for pirlimycin (I) residue in bovine milk and liver have been developed based on HPLC-thermospray (TSP) MS. Milk sample preparation consisted of precipitating the milk proteins with acidified acetonitrile followed by a solvent partitioning with a mixture of n-butyl chloride and hexane, extraction of I from the aqueous phase into methylene chloride (MC), and solid-phase extraction clean-up. For liver, samples (2 g) were extracted with 0.25% trifluoroacetic acid in acetonitrile. The aqueous component was released from the organic solvent with n-butyl chloride. The aqueous solution was reduced in volume by evaporation, basified with ammonium hydroxide, then extracted with MC. The MC was evaporated to dryness and the dried residue reconstituted in 2.0 ml of 0.1 M ammonium acetate for analysis. A chromatographically resolved stereoisomer of I with TSP-MS response characteristics identical to I was used as an internal standard (I.S.) for quantitative analysis based on the ratio of peak areas of I to I.S. in the protonated molecular-ion chromatogram at m/z 411.2.The method for milk was validated by the analysis of control milk samples spiked with I at concentrations from 0.05 to 0.8 μg/ml. The overall recovery of pirlimycin across this concentration range was 95.4% ± 8.7%. The limit of quantitation (LOQ) and limit of confirmation (LOC) of the method were validated to be 0.05 μg/ml and 0.10 μg/ml, respectively.The method for liver was validated by the analysis of control liver samples spiked with I at concentrations ranging from 0.025 to 1.0 μg/g. The overall recovery of pirlimycin was 97.6% ± 5.1% in this concentration range. The validated limit of quantitation (LOQ) and limit of confirmation (LOC) of the method were 0.025 μg/g and 0.10 μg/g, respectively.Four diagnostic ions for I were monitored for confirmation: the pseudo-molecular ions (M + H)⁺ at m/z 411.2 (³⁵Cl) and m/z 413.2 (³⁷Cl), and fragment ions at m/z 375.2 and 158.1. Confirmatory criteria were defined for these assays.     

High-performance liquid chromatography-thermospray mass spectrometry of 5,6-dihydroxyeicosatrienoate-1,5-lactone from tissue homogenates

We have developed a method for the analysis of 5,6-dihydroxyeicosatrienoate-1,5-lactone (5,6-DiHETriE-δ-lactone) in tissue homogenates, supplemented with NADPH and arachidonic acid [20:4(n−6)] as a substrate. During the incubation and the extraction, most of the 5,6-epoxyeicosatrienoic acid (5,6-EpETriE) was converted to 5,6-dihydroxyeicosatrienoic acid (5,6-DiHETriE), and most of the 5,6-DiHETriE was converted to 5,6-DiHETriE-δ-lactone. Consequently, the chief degradation product of 5,6-EpETriE and 5,6-DiHETriE in the incubation mixture was 5,6-DiHETriE-δ-lactone. 5,6-DiHETriE-δ-lactone, corresponding to [20:4(n−6)], was shown to be characterized by a high intensity of quasimolecular ions (MH⁺ and MNH₄⁺), using ion analysis obtained by reversed-phase HPLC-thermospray MS. On selected-ion monitoring (SIM) chromatograms of 5,6-DiHETriE-δ-lactone and with deuterium-labeled 15(s)-hydroxyeicosatetraenoic acid as the internal standard, the regression equation of the peak-area ratio and the amount of 5,6-DiHETriE-δ-lactone was y = 12.2x + 0.7 (r = 0.9996). 5,6-Epoxygenase activity was represented as the sum of the amount of 5,6-DiHETriE-δ-lactone, 5,6-EpETriE and 5,6-DiHETriE per mg protein, after 30 min in an incubation mixture. The activity from rat brain homogenate decreased considerably with growth of the rat.     

Simultaneous quantitative determination method for ceramide species from crude cellular extracts by high-performance liquid chromatography-thermospray mass spectrometry

I have developed a simple method which enabled simultaneous analysis of ceramides in the subcellular fractions from cultured cells by HPLC-thermospray mass spectrometry. The HPLC-thermospray mass spectra from ceramide standards were characterized by the high intensity of the MNa(+) and MH(+)-H(2)O ions. As the other minor ions, MK(+), MH(+) and m/z 282 ions were detected. Although the preponderance of MNa(+) ions compared with the MH(+)-H(2)O ions was detected in non-hydroxy fatty acid-ceramides, the preponderance of MH(+)-H(2)O ions based on the elimination of the hydroxyl group introduced at the alpha-position of acyl-portion compared with the MNa(+) ions was detected in alpha-hydroxy fatty acid-ceramides. In calibrations for authentic ceramides using N-octanoylsphingosine as an internal standard, an approximately linear relationship existed between the ratios of peak-areas of each ceramide to that of the internal standard and the known amounts of each ceramide. The factor (f) of each ceramide was calculated as follows; N-oleoyl-D-sphingosine (f=0.45), N-palmitoyl-D-sphingosine (f=0.40), N-stearoyl-D-sphingosine (f=0.39), N-nervonoyl-D-sphingosine (f=0.39) and N-lignoceroyl-D-sphingosine (f=0.35). In subcellular fractions from A549 and HepG2 cells, although ceramide species content per mg protein was high in the nuclear envelope fractions, the 7000 g pellet fractions and the 100000 g pellet fractions, a large portion of the ceramide species was concentrated in the nuclear envelope fraction. In addition, this method was applied to a mild alkaline hydrolyzate of total ceramides from pig stratum corneum, and MNa(+)/MH(+)-H(2)O ions corresponding to several omega-hydroxyacyl-ceramides were detected.     

Direct analysis of microbial extracts containing metabolites of ethylenediamine-type antihistamines via high-performance liquid chromatography-thermospray mass spectrometry

The utility of high-performance liquid chromatography-thermospray mass spectrometry (HPLC-TSMS) for the characterization of the ethylenediamine-type antihistamines, pyrilamine, methapyrilene, tripelennamine, and thenyldiamine, and their methylene chloride-extractable microbial metabolites from a biological matrix is demonstrated. Typically, the [M + H]+ ion was detected as the base peak in the TS mass spectra of these compounds. The ethylenediamine-type antihistamine metabolites were detected in an extract of a fungal culture grown in the presence of 5 mg of the antihistamine. A detection limit of 200 ng was observed for the HPLC-TSMS analysis of pyrilamine.     

Determination of the active metabolite of sibutramine by liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and specific method for the determination of the active primary amine metabolite of sibutramine, N-di-desmethylsibutramine (BTS 54,505), in human plasma was developed, based on high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (MS-MS). The samples were extracted from plasma with methyl tert.-butyl ether, followed by separation and evaporation after addition of the internal standard, propranolol, and basification with sodium hydroxide. The residue was reconstituted in mobile phase and injected into the HPLC-MS-MS system. Chromatography was performed on an ODS MS column with a mobile phase consisting of acetonitrile (containing 0.1% trifluoroacetic acid, v/v)-0.1% trifluoroacetic acid (55:45, v/v) at a flow-rate of 0.3 ml/min. Multiple reaction monitoring using precursor-->product ion combinations at m/z 252.00-->125.00 and 260.00-->115.70 was applied to determine BTS 54,505 and propranolol, respectively. Linearity was confirmed in the concentration range 0.328-32.8 ng/ml in human plasma and the imprecision of this assay was less than 19.90% over the entire concentration range. The method is sufficiently sensitive and repeatable to be used in pharmacokinetic studies.     

Determination of the active metabolite of prulifloxacin in human plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric method (LC-MS/MS) for the determination of ulifloxacin, the active metabolite of prulifloxacin, in human plasma is described. After sample preparation by protein precipitation with methanol, ulifloxacin and ofloxacin (internal standard) were chromatographically separated on a C(18) column using a mobile phase consisting of methanol, water and formic acid (70:30:0.2, v/v/v) at a flow rate of 0.5 ml/min and then were detected using MS/MS by monitoring their precursor-to-product ion transitions, m/z 350-->m/z 248 for ulifloxacin and m/z 362-->m/z 261 for ofloxacin, in selected reaction monitoring (SRM) mode. Positive electrospray ionization was used for the ionization process. The linear range was 0.025-5.0 microg/ml for ulifloxacin with a lower limit of quantitation of 0.025 microg/ml. Within- and between-run precision was less than 6.6 and 7.8%, respectively, and accuracy was within 2.0%. The recovery ranged from 92.1 to 98.2% at the concentrations of 0.025, 0.50 and 5.0 microg/ml. Compared with the reported LC method, the present LC-MS/MS method can directly determine the ulifloxacin in human plasma without any need of derivatization. The present method has been successfully used for the pharmacokinetic studies of a prulifloxacin formulation product after oral administration to healthy volunteers.     

Determination of cryptotanshinone and its metabolite in rat plasma by liquid chromatography-tandem mass spectrometry

A sensitive and selective LC-MS-MS method has been developed and validated for the determination of cryptotanshinone (CTS) and its active metabolite tanshinone II A (TS II A) in rat plasma using fenofibrate (FOFB) as internal standard. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved on a Waters symmetry ODS column using methanol and water (85:15) as mobile phase delivered at 1.0 mL/min. LC-MS-MS analysis was carried out on a Finnigan LC-TSQ Quantum mass spectrometer using atmospheric pressure chemical ionization (APCI) and positive multiple reaction monitoring. Ions monitored were m/z 297.0--> 251.0 for CTS, m/z 295.0--> 249.0 for TS II A, and m/z 361.1--> 233.0 for FOFB with argon at a pressure of 0.2 Pa and collision energy of 25 eV for collision-induced dissociation (CID). The assay was linear over the range 0.1-20 ng/mL for CTS and 0.2-15 ng/mL for TS II A. The average recoveries of CTS and TS II A from rat plasma were 93.7 and 94.7%, respectively. The established method has been applied in a pharmacokinetic study of CTS in rats.     

Determination of trenbolone and its metabolite in bovine fluids by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method has been developed for the determination of trenbolone in bovine urine and serum. The aim was a control of the misuse of trenbolone in food-producing animals. The procedure involved, in both cases, a preliminary solid-phase clean-up followed by a liquid-liquid extraction for urine samples after a preliminary enzymatic hydrolysis. The extracts have been directly analysed by reversed-phase LC-MS-MS in selected reaction monitoring (SRM), acquiring two diagnostic product ions from the chosen precursor [M+H](+). The procedures were validated across the concentration range of 1-1500 ng/ml. The linearity, the inter- and intra-day accuracy and precision have been determined. The procedure was specific and the accuracy values were better than 20% at the limit of quantitation of spiked samples. The limit of quantification (LOQ) and the limit of detection (LOD) were, respectively, 1 ng/ml and 350 pg/ml for urine and serum. According to the draft, SANCO/1805/2000, we determined the decision limit CCalpha and the detection capability CCbeta. The recovery values for urine ranged from 87 to 128%, and for plasma the recovery was 70+/-4%. The procedure proved to be simple and suitable for routine and confirmatory purposes such as those developed for residue studies.     

Simultaneous determination of JTT-501 and its main metabolite in human plasma by liquid chromatography-ionspray mass spectrometry

An LC-MS-MS analytical method was developed for the determination of a new antidiabetic agent, JTT-501 and its main metabolite (JTP-20604) in human plasma. The compounds were isolated from plasma by protein precipitation before analysis by HPLC with atmospheric pressure positive ionisation MS-MS detection. An isotopically labelled analog of JTT-501 was used as the internal standard. Linearity was demonstrated over the calibration range of about 5-10000 ng/ml for both compounds. The assay was validated with respect to accuracy, precision and analyte stability. This method was used for the determination of plasma concentrations for the two compounds in a clinical tolerability study. A cross-validation exercise between two different mass spectrometers, used for the determination of clinical samples, is also reported.     

High-throughput screening of corticosteroids and basic drugs in horse urine by liquid chromatography-tandem mass spectrometry

This paper describes two high-throughput liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods for the screening of two important classes of drugs in equine sports, namely corticosteroids and basic drugs, at low ppb levels in horse urine. The method utilized a high efficiency reversed-phase LC column (3.3 cm L x 2.1 mm i.d. with 3 microm particles) to provide fast turnaround times. The overall turnaround time for the corticosteroid screen was 5 min and that for the basic drug screen was 8 min, inclusive of post-run and equilibration times. Method specificity was assessed by analysing a total of 35 negative post-race horse urine samples. No interference from the matrices at the expected retention times of the targeted masses was observed. Inter-day precision for the screening of 19 corticosteroids and 48 basic drugs were evaluated by replicate analyses (n = 10) of a spiked sample on 4 consecutive days. The results demonstrated that both methods have acceptable precision to be used on a routine basis. The performance of these two methods on real samples was demonstrated by their applications to drug administration and positive post-race urine samples.     

Simultaneous determination of ten antiarrhythic drugs and a metabolite in human plasma by liquid chromatography--tandem mass spectrometry

A simple, accurate and selective LC-MS/MS method was developed and validated for simultaneous quantification of ten antiarrhythic drugs (diltiazem, amiodarone, mexiletine, propranolol, sotalol, verapamil, bisoprolol, metoprolol, atenolol, carvedilol) and a metabolite (norverapamil) in human plasma. Plasma samples were simply pretreated with acetonitrile for deproteinization. Chromatographic separation was performed on a Capcell C(18) column (50mmx2.0mm, 5microm) using a gradient mixture of acetonitrile and water (both containing 0.02% formic acid) as a mobile phase at flow rate of 0.3ml/min. The analytes were protonated in the positive electrospray ionization (ESI) interface and detected in multiple reaction monitoring (MRM) mode. Calibration curves were linear over wide ranges from sub- to over-therapeutic concentration in plasma for all analytes. Intra- and inter-batch precision of analysis was <12.0%, accuracy ranged from 90% to 110%, average recovery from 85.0% to 99.7%. The validated method was successfully applied to therapeutic drug monitoring (TDM) of antiarrhythic drugs in routine clinical practice.     

Simultaneous determination of amodiaquine and its active metabolite in human blood by ion-pair liquid chromatography-tandem mass spectrometry

A sensitive and selective ion-pair liquid chromatography-tandem mass spectrometric method (IP-LC-MS/MS) for the simultaneous determination of amodiaquine (AQ) and its active metabolite, N-desethylamodiaquine (AQm), in human blood has been developed and validated. Pentafluoropropionic acid (PFPA) was applied as ion-pairing reagent in reversed-phase chromatographic separation. The effects of PFPA concentrations and the volume fraction of acetonitrile in the mobile phase on the retention of analytes were investigated on a Venusil MP-C(18) column, and the mobile phase was finally optimized as acetonitrile:water (23:77, v/v) with 0.0667% PFPA in the aqueous phase. The results proved that PFPA as an ion-pairing reagent could provide desirable chromatographic performance in the IP-LC-MS/MS determination of 4-aminoquinoline compounds. Blood samples were protein precipitated with acetonitrile using hydroxychloroquine (OHCQ) as the internal standard. The detection was carried out in multiple reaction monitoring (MRM) mode via positive atmospheric pressure chemical ionization (APCI) interface. The lower limits of quantification were established at 0.150 and 1.50 ng/mL for AQ and AQm, respectively. The validated IP-LC-MS/MS method was applied to a clinical pharmacokinetic study of AQ and AQm in human blood after an oral administration of 600 mg AQ hydrochloride (45 9mg base).     

Assay of zofenopril and its active metabolite zofenoprilat by liquid chromatography coupled with tandem mass spectrometry

Zofenopril is a pro-drug designed to undergo metabolic hydrolysis yielding the active free sulfhydryl compound zofenoprilat, which is an angiotensin converting enzyme (ACE) inhibitor, endowed also with a marked cardioprotective activity. A simple, highly sensitive specific LC–MS–MS method was developed for the determination of zofenopril and zofenoprilat in human plasma. In order to prevent oxidative degradation of zofenoprilat and its internal standard, their free sulfhydryl groups were protected by treatment with N-ethylmaleimide (NEM), which produced the succinimide derivatives. The compounds and their corresponding fluorine derivatives, used as internal standards, were extracted from plasma with toluene. The reconstituted dried extracts were chromatographed and then monitored by a triple-stage-quadrupole instrument operating in the negative ion spray ionization mode. The method was validated over the concentration range of 1–300 ng/ml for zofenopril and 2–600 ng/ml for zofenoprilat. Inter- and intra-assay precision and accuracy of both zofenopril and zofenoprilat were better than 10%. The limit of quantitation was 1 ng/ml with zofenopril and 2 ng/ml with zofenoprilat. Extraction recovery proved to be on average 84.8% with zofenopril and 70.1% with zofenoprilat. Similar recoveries were shown by the above two internal standards. The method was applied to measure plasma concentrations of zofenopril and zofenoprilat in 18 healthy volunteers treated orally with zofenopril calcium salt at the dose of 60 mg.     

Drug metabolite profiling and identification by high-resolution mass spectrometry

Mass spectrometry plays a key role in drug metabolite identification, an integral part of drug discovery and development. The development of high-resolution (HR) MS instrumentation with improved accuracy and stability, along with new data processing techniques, has improved the quality and productivity of metabolite identification processes. In this minireview, HR-MS-based targeted and non-targeted acquisition methods and data mining techniques (e.g. mass defect, product ion, and isotope pattern filters and background subtraction) that facilitate metabolite identification are examined. Methods are presented that enable multiple metabolite identification tasks with a single LC/HR-MS platform and/or analysis. Also, application of HR-MS-based strategies to key metabolite identification activities and future developments in the field are discussed.     

Identification of a novel selenium metabolite, Se-methyl-N-acetylselenohexosamine, in rat urine by high-performance liquid chromatography--inductively coupled plasma mass spectrometry and--electrospray ionization tandem mass spectrometry

The major urinary metabolite of selenium (Se) in rats was identified by HPLC-inductively coupled argon plasma mass spectrometry (ICP-MS) and--electrospray tandem mass spectrometry (ESI-MS/MS). As the urine sample was rich in matrices such as sodium chloride and urea, it was partially purified to meet the requirements for ESI-MS. The group of signals corresponding to the Se isotope ratio was detected in both the positive and negative ion modes at m/z 300 ([M+H]+) and 358 ([M+CH3COO]-) for 80Se, respectively. These results suggested that the molecular mass of the Se metabolite was 299 Da for 80Se. The Se metabolite was deduced to contain one methylselenyl group, one acetyl group and at least two hydroxyl groups from the mass spectra of the fragment ions. The spectrum of the Se metabolite was completely identical to that of the synthetic selenosugar, 2-acetamide-1,2-dideoxy-beta-D-glucopyranosyl methylselenide. However, the chromatographic behavior of the Se metabolite was slightly different from that of the synthetic selenosugar. Thus, the major urinary Se metabolite was assigned as a diastereomer of a selenosugar, Se-methyl-N-acetyl-selenohexosamine.     

Mediator lipidomics by liquid chromatography-tandem mass spectrometry

Comprehensive quantitative analysis of lipid mediators using liquid chromatography-tandem mass spectrometry is an effective strategy in the elucidation of disease mechanisms; but technically, it has been and is still a great challenge to achieve reliable datasets that cover variety of lipid metabolites contained at trace levels in complex biological matrices. In this opinion article, we introduce our experiences in developing lipid mediator profiling systems, and deliver some comments on limitations of current methodology.     

Urinary antihypertensive drug metabolite screening using molecular networking coupled to high-resolution mass spectrometry fragmentation

Introduction

Mass spectrometry is the current technique of choice in studying drug metabolism. High-resolution mass spectrometry in combination with MS/MS gas-phase experiments has the potential to contribute to rapid advances in this field. However, the data emerging from such fragmentation spectral files pose challenges to downstream analysis, given their complexity and size.

Objectives

This study aims to detect and visualize antihypertensive drug metabolites in untargeted metabolomics experiments based on the spectral similarity of their fragmentation spectra. Furthermore, spectral clusters of endogenous metabolites were also examined.

Methods

Here we apply a molecular networking approach to seek drugs and their metabolites, in fragmentation spectra from urine derived from a cohort of 26 patients on antihypertensive therapy. The mass spectrometry data was collected on a Thermo Q-Exactive coupled to pHILIC chromatography using data dependent analysis (DDA) MS/MS gas-phase experiments.

Results

In total, 165 separate drug metabolites were found and structurally annotated (17 by spectral matching and 122 by classification based on a clustered fragmentation pattern). The clusters could be traced to 13 drugs including the known antihypertensives verapamil, losartan and amlodipine. The molecular networking approach also generated clusters of endogenous metabolites, including carnitine derivatives, and conjugates containing glutamine, glutamate and trigonelline.

Conclusions

The approach offers unprecedented capability in the untargeted identification of drugs and their metabolites at the population level and has great potential to contribute to understanding stratified responses to drugs where differences in drug metabolism may determine treatment outcome.

     

Quantitation of the flavonoid wogonin and its major metabolite wogonin-7 beta-D-glucuronide in rat plasma by liquid chromatography-tandem mass spectrometry

This study described the application of liquid chromatography-tandem mass spectrometry for the quantitation of wogonin and its major metabolite in rat plasma. Only one conjugated metabolite with glucuronic acid was identified by chromatographic and electrospray multi-stage mass spectrometric assay. A derivatization reaction with 2-chlorethanol further demonstrated that the metabolite was wogonin-7 beta-D-glucuronide (W-7-G), not wogonin-5 beta-D-glucuronide. Other conjugated metabolites, e.g., sulfates and glucosides, were not detected. The plasma concentration of free wogonin was determined using atmospheric pressure chemical ionization source in the selected reaction monitoring mode. The method had a lower limit of quantitation of 0.25 ng/ml for wogonin, which offered increased sensitivity, selectivity and speed of analysis over an existing method. Incubation of the plasma samples with beta-glucuronidase allows the quantitation of W-7-G. This quantitation method was successfully applied to a preclinical pharmacokinetic study of wogonin and its major metabolite, W-7-G, after an oral administration of 5 mg/kg wogonin to rats.     

Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry

Modeling of metabolic networks as part of systems metabolic engineering requires reliable quantitative experimental data of intracellular concentrations. The hydrophilic interaction liquid chromatography–electrospray ionization–tandem mass spectrometry (HILIC–ESI–MS/MS) method was used for quantitative profiling of more than 50 hydrophilic key metabolites of cellular metabolism. Without prior derivatization, sugar phosphates, organic acids, nucleotides, and amino acids were measured under alkaline and acidic mobile phase conditions with pre-optimized multiple reaction monitoring (MRM) transitions. Irrespective of the polarity mode of the acquisition method used, alkaline conditions achieved the best quantification limits and linear dynamic ranges. Fully 90% of the analyzed metabolites presented detection limits better than 0.5 pmol (on column), and 70% presented 1.5-fold higher signal intensities under alkaline mobile phase conditions. The quality of the method was further demonstrated by absolute quantification of selected metabolites in intracellular extracts of Escherichia coli. In addition, quantification bias caused by matrix effects was investigated by comparison of calibration strategies: standard-based external calibration, isotope dilution, and standard addition with internal standards. Here, we recommend the use of alkaline mobile phase with polymer-based zwitterionic hydrophilic interaction chromatography (ZIC–pHILIC) as the most sensitive scenario for absolute quantification for a broad range of metabolites.