Application of Cookson-type reagents for biomedical HPLC and LC/MS analyses: a brief overview.

High-performance liquid chromatography (HPLC), LC/mass spectrometry (MS) and LC/tandem mass spectrometry (MS/MS) have been widely used for biomedical analyses, in which chemical derivatization is one of the most important methods to increase the sensitivity and selectivity. A Cookson-type reagent [4-substituted-1,2,4-triazoline-3,5-dione (4-substituted-TAD)] reacts with the compound bearing a conjugated diene, such as the vitamin D compound, to quantitatively form the stable Diels-Alder adduct. The reagent with a chromophore or fluorophore at the 4-position of TAD yields a highly responsive adduct for the UV or fluorescence detection, respectively. The Diels-Alder adduct with a Cookson-type reagent having a permanently charged, proton-affinitive or electron-affinitive moiety is sensitively detected by a specific MS analyzer. This paper is a brief overview of the applications of the reagents for biomedical analyses mainly using HPLC or LC/MS(/MS).

A specific LC/ESI-MS/MS method for determination of 25-hydroxyvitamin D3 in neonatal dried blood spots containing a potential interfering metabolite, 3-epi-25-hydroxyvitamin D3.

Vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes in later life. A method for the quantification of 25-hydroxyvitamin D(3) [25(OH)D(3), the best-established indicator of vitamin D status] in neonatal dried blood spots (DBSs) using LC/ESI-MS/MS has been developed and validated. The method employed two steps of derivatization, a Diels-Alder reaction with 4-phenyl-1,2,4-triazoline-3,5-dione followed by acetylation, to enhance the detectability of 25(OH)D(3) in ESI-MS/MS and to separate 25(OH)D(3) from 3-epi-25-hydroxyvitamin D(3) [3-epi-25(OH)D(3)], a potent interfering metabolite. 25(OH)D(3) was extracted from two DBS punches (3 mm in diameter, equivalent to 5.3 µL of whole blood), purified using an Oasis HLB(®) cartridge, and subjected to derivatization prior to analysis with LC/ESI-MS/MS. 25-Hydroxyvitamin D(4) was used as the internal standard. This method was reproducible (intra- and inter-assay RSDs, <6.9%) and accurate (analytical recovery, 95.2-102.7%), and the LOQ was 3.0 ng/mL. The developed method enabled specific quantification of 25(OH)D(3) in neonatal DBSs and detection of vitamin D deficiency without interference from 3-epi-25(OH)D(3).

Salivary chenodeoxycholic acid and its glycine-conjugate: their determination method using LC-MS/MS and variation of their concentrations with increased saliva flow rate.

Measurement of steroid levels in saliva has been proposed as a new laboratory tool for characterizing steroid metabolism, but it is not known whether the salivary levels of bile acids can be measured with accuracy and if so, whether such measurements provide information that is of clinical value. We developed and validated a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the quantification of chenodeoxycholic acid (CDCA) and glycochenodeoxycholic acid (GCDCA), representative primary non-amidated and glycine-conjugated bile acids, in whole saliva. We also examined whether the salivary bile acid concentrations were dependent on the saliva flow rate, because this is a very important aspect in a discussion of the utility of salivary diagnostics. Saliva was deproteinized with ethanol and purified using a Strata-X cartridge. Bile acids were converted to their hydrazide derivatives using 2-hydrazinopyridine, and subjected to LC-MS/MS. Quantification was based on selected reaction monitoring using characteristic transitions, and deuterated CDCA and GCDCA were used as internal standards. This method allowed the reproducible and accurate quantification of the salivary bile acids using a 200-microl sample and the limits of quantification for CDCA and GCDCA were 25 and 50pg/ml, respectively. Using this method, the effect of increased saliva flow rate by gum-chewing on the salivary concentrations of CDCA and GCDCA was determined. The salivary level of GCDCA was significantly decreased by gum-chewing, whereas the concentration of CDCA remained constant. These results indicate that there is a good possibility that saliva may be a clinical tool for non-amidated bile acid testing.

Advances in determination of vitamin D related compounds in biological samples using liquid chromatography-mass spectrometry: a review.

The measurements of the serum/plasma concentrations of vitamin D metabolites are widely used for the diagnostic assessment and follow-up of several diseases, such as chronic renal failure and osteoporosis. These metabolites have usually been measured by protein binding assays, such as radioimmunoassay and radioreceptor assay. Although these techniques will doubtless continue to be the methods of choice for routine use in the clinical field, their specificity and accuracy are sometimes poor due to interference from other metabolites and lipids. Among the alternative methods, liquid chromatography (LC) coupled with mass spectrometry (MS) has been used for the analysis of these metabolites and even synthetic vitamin D analogues (therapeutic agents) due to its sensitivity and selectivity. This article reviews recent advances in the determination of vitamin D metabolites and related compounds in biological samples using LC-MS.

Studies on neurosteroids XXVI. Fluoxetine-evoked changes in rat brain and serum levels of neuroactive androgen, 5 alpha-androstane-3 alpha,17 beta-diol.

It is well known that fluoxetine (Fluox), a selective serotonin reuptake inhibitor, increases the brain content of allopregnanolone (AP), a potent neuroactive steroid that positively modulates the action of gamma-aminobutyric acid (GABA) at the GABA type A receptors, but the influence of Fluox on the brain and serum levels of a neuroactive androgen, 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-Adiol), is poorly understood. In this study, we examined the Fluox-evoked changes in the 3alpha,5alpha-Adiol levels and compared the level changes of 3alpha,5alpha-Adiol with those of AP. The brain and serum 3alpha,5alpha-Adiol and AP levels were determined using previously developed LC-MS/MS. The ratio of the brain 3alpha,5alpha-Adiol to the serum 3alpha,5alpha-Adiol concentrations (B/S value) was significantly elevated in the rats intraperitoneally administered Fluox (10 mg/kg). Although the magnitude of the Fluox-evoked level change in 3alpha,5alpha-Adiol was much lower than that in AP, this study demonstrated that the 3alpha,5alpha-Adiol content is also influenced by Fluox. The most probable cause for the increase in the B/S value by the Fluox treatment is the activation of the 3alpha-hydroxysteroid dehydrogenase enzyme followed by the promotion of the de novo biosynthesis of 3alpha,5alpha-Adiol in the brain.

Analyses of biologically active steroids: antitumor active OSW-1 and cardiotonic marinobufotoxin, by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem mass spectrometry.

Naturally occurring constituents of biological or pharmaceutical interest often exist in the form of glycosides or conjugates. Mass spectral investigations of these compounds require soft ionization techniques if information on molecular mass, sugar sequence, or conjugate content is desired. In this study, matrix-assisted laser desorption/ionization (MALDI) quadrupole ion trap (QIT) time-of-flight tandem mass spectrometry (TOF-MS(n)) was used to identify both OSW-1, an acetylated cholestane diglycoside showing antitumor activity, and the cardiotonic steroid, bufotoxin. Each molecular-related ion was identified, and subsequent collision-induced dissociation experiments in which a molecular-related ion was selected as a precursor ion produced the characteristic product ions that are essential for structural elucidation. OSW-1 and its analogue with a modified side chain, thienyl OSW-1, were synthesized, and bufotoxins, i.e., marinobufotoxin and its homologue, marinobufagin 3-pimeloylarginine ester, were isolated from toad venom. On MALDI-TOF-MS, sodium-adduct [M+Na](+) ions were observed in the steroid glycosides, although protonated [M+H](+) ions were relatively more abundant than sodium-adduct [M+Na](+) ions in the bufotoxins. On the basis of tandem MS results, we propose key fragmentation pathways. The sugar moiety or side chain from the precursor ion was eliminated in OSW-1. However, characteristic product ions originating from the cleavage of the side chain with an ester formation were observed in the bufotoxins. Post-source decay (PSD) on MALDI-TOF-MS is also described when evaluating alpha-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid as a matrix to obtain useful ions required for the identification of compound.

Simultaneous determination of salivary testosterone and dehydroepiandrosterone using LC-MS/MS: Method development and evaluation of applicability for diagnosis and medication for late-onset hypogonadism.

Late-onset hypogonadism (LOH) is a male-specific disorder caused by the age-related decline in androgens, such as testosterone (T). A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the simultaneous quantification of T and its precursor, dehydroepiandrosterone (DHEA), in human saliva has been developed and validated. The saliva was deprotenized with acetonitrile, purified using a Strata-X cartridge, derivatized with 2-hydrazino-1-methylpyridine, and subjected to LC-MS/MS. The recovery rates of the steroids during the pretreatment were about 90%. Quantification was based on selected reaction monitoring using characteristic transitions, and deuterated T and DHEA were used as internal standards. This method allowed the reproducible (inter- and intra-assay precisions, <2.9%) and accurate (accuracy, 98.5-101.8%) quantification of the salivary androgens using a 500-microl sample and the limits of quantification for both androgens were 10 pg/ml. As preliminary steps in the practical application of the developed method in diagnosis and medication for LOH, the diurnal rhythms, inter-day alternations and age differences in the salivary T and DHEA were examined; the method found that the salivary T and DHEA show specific diurnal rhythms, significant alternations in early morning and pronouncedly decline with age. The method also enabled the determination of the changes in the individual T and DHEA levels after the DHEA supplementation, which is expected to be a new and easy medication for LOH. Thus, the developed method has satisfactory applicability in the diagnosis and medication for LOH.

Studies on neurosteroids XXV. Influence of a 5alpha-reductase inhibitor, finasteride, on rat brain neurosteroid levels and metabolism.

In this study, we examined the influence of finasteride (FIN), a 5alpha-reductase inhibitor, on the brain levels and metabolism of neurosteroids [allopregnanolone (AP), 3alpha-dihydroprogesterone (3alpha-DHP), progesterone (PROG), 20alpha-dihydroprogesterone and 11-deoxycorticosterone (DOC)] in rats exposed to immobilization stress. For this purpose, the sensitive, reproducible and accurate liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) methods that enable the quantification of trace amounts of brain neurosteroids were first developed. The animal study using these methods demonstrated that FIN dose-dependently inhibits the stress-induced elevation of the brain AP, a potent positive modulator of the gamma-aminobutyric acid (GABA) type A receptors, and a 10 mg/kg dose of FIN can almost completely deplete AP in the brains. The study also found that the 20alpha-reduction of PROG is enhanced when its 5alpha-reduction pathway is inhibited in the brains. No change was found in the brain levels of 3alpha-DHP, another GABAergic neurosteroid, and DOC by the administration of FIN.

Studies on neurosteroids XX. Liquid chromatography-tandem mass spectrometric method for simultaneous determination of testosterone and 5alpha-dihydrotestosterone in rat brain and serum.

Testosterone (T) and 5alpha-dihydrotestosterone (DHT) are now referred to not only as androgenic steroid hormones, but also as neuroactive steroids, because they elicit anesthetic and anxiolytic effects. Methods using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) coupled with derivatization are developed and validated to examine rat brain and serum levels of T and DHT and their stress-induced changes. The steroids are extracted with methanol-acetic acid from the brain tissue or serum, purified using solid-phase extraction cartridges, derivatized with a permanently charged reagent, 2-hydrazino-1-methylpyridine, and subjected to LC-MS-MS. [19,19,19-(2)H3]-T is used as the internal standard. The intra- and inter-assay coefficients of variation are below 10.0%, and the analytical recoveries are 98.1-103.0%. The developed methods are applied to the animal study and it was found that a fair amount of DHT is continuously and locally synthesized in the brain, and its level is not changed by the immobilization stress and depends on the brain T level.

Characterization of in vivo metabolites of toad venom using liquid chromatography-mass spectrometry.

The structures of in vivo metabolites of marinobufotoxin (marinobufagin 3-suberoylarginine ester), marinobufagin, or bufalin which are typical components of toad venom widely used as the traditional Chinese drug, Ch'an Su, are confirmed using authentic samples based on their liquid chromatography-mass spectrometric behavior. A rat is orally administered 2 mg of the previously mentioned components of toad venom, the serum is collected 30 min after the administration, extracted, and then characterized. Marinobufotoxin is hydrolyzed and further epimerized into 3-epimarinobufagin, but marinobufagin 3-hemisuberate is not detected. After the administration of marinobufagin, 3-epimarinobufagin is detected in both the male and female rats, but marinobufagin 3-sulfate is formed only in the female rats. Bufalin is metabolized to 3-epibufalin, which is found to undergo further conjugation resulting in its 3-glucuronide. Furthermore, 3-epibufalin 3-sulfate is formed only in the female rats.

Studies on neurosteroids XXIV. Determination of neuroactive androgens, androsterone and 5alpha-androstane-3alpha,17beta-diol, in rat brain and serum using liquid chromatography-tandem mass spectrometry.

The development and validation of liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) methods that enable the quantification of neuroactive androgens, androsterone (5alpha-androstan-3alpha-ol-17-one, 3alpha,5alpha-A) and 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-Adiol), in the rat brain and serum are presented. The androgens were extracted with methanol-acetic acid, purified using solid-phase extraction cartridges, derivatized with an ESI-active reagent, isonicotinoyl azide (INA), and then subjected to LC-ESI-MS/MS. The quantifications were based on selected reaction monitoring mode using the characteristic transitions of the INA derivatives. The methods allowed the reproducible and accurate quantification of the brain and serum neuroactive androgens using a 100 mg or 100 microL sample; the intra- and inter-assay relative standard deviations were below 3.6%, and the percentage accuracy values were 97.1-103.7% for both androgens. The animal study using the methods suggests that most of 3alpha,5alpha-Adiol found in the brain is derived from the periphery, while 3alpha,5alpha-A is not only transported from the periphery into the brain, but also synthesized in the brain by the oxidation of 3alpha,5alpha-Adiol. The androgens in the rats intraperitoneally administered finasteride, a 5alpha-reductatse inhibitor, were also measured; this treatment significantly reduced the brain 3alpha,5alpha-A and 3alpha,5alpha-Adiol levels and increased only the brain level of androstenedione, the precursor of 3alpha,5alpha-A.

Simultaneous determination of 17alpha-hydroxypregnenolone and 17alpha-hydroxyprogesterone in dried blood spots from low birth weight infants using LC-MS/MS.

17alpha-hydroxypregnenolone (17OHPreg) has heretofore been considered to be the major cause of the false elevated 17alpha-hydroxyprogesterone (17OHP) value in the immunoassay-based newborn screening for congenital adrenal hyperplasia (CAH). To verify this point, we developed a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method that enables the simultaneous quantification of 17OHPreg and 17OHP in the dried blood filter papers and measured their blood levels in infants, especially in infants with low birth weights. Steroids were extracted from the filter papers with methanol, purified using a Strata-X cartridge, derivatized with 2-hydrazinopyridine and subjected to LC-MS/MS. Validation tests proved that this method was specific and reproducible; endogenous steroids did not interfere with the quantifications, and the intra- and inter-assay coefficients of variation were below 5.2%. The limits of quantitation were 1.0 and 0.5 ng/mL for 17OHPreg and 17OHP, respectively, when 3 disks (3 mm in diameter) of the filter papers (corresponding to 8 microL of whole blood) were used. The blood 17OHPreg level was elevated in the very low birth weight (1000-1500 g) infants and extremely low birth weight (<1000 g) infants, compared to those in the normal birth weight (>2500 g) infants (P<0.05). However, the 17OHPreg concentration was not high enough to cause the false positive results in the enzyme immunoassay-based screening, and it was considered that the false positive results come from other endogenous components rather than 17OHPreg.

Liquid chromatography-tandem mass spectrometric method for determination of salivary 17alpha-hydroxyprogesterone: a noninvasive tool for evaluating efficacy of hormone replacement therapy in congenital adrenal hyperplasia.

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS-MS) method for the quantification of 17alpha-hydroxyprogesterone (17OHP) in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with a highly proton-affinitive reagent, 2-hydrazinopyridine, and subjected to LC-MS-MS. Quantification was based on the selected reaction monitoring, and deuterated 17OHP was used as the internal standard. This method allowed the reproducible and accurate quantification of the salivary 17OHP using a 200-mul sample, and the limit of quantitation was 5.0 pg/ml. The developed method was applied to clinical studies. A linear relationship was found to be positive (r(2)=0.975) between the blood 17OHP level and the salivary 17OHP level measured using the proposed method. The result from the salivary 17OHP measurement in patients with congenital adrenal hyperplasia demonstrated that the proposed method is very useful for monitoring of the therapeutic efficacy during hormone replacement therapy.

Liquid chromatography-tandem mass spectrometric method for the determination of salivary 25-hydroxyvitamin D3: a noninvasive tool for the assessment of vitamin D status.

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the determination of 25-hydroxyvitamin D(3) [25(OH)D(3)] in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with a Cookson-type reagent, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and subjected to LC-MS/MS. The PTAD derivative was much more easily ionized in positive-ESI-MS and efficiently produced a characteristic product ion during MS/MS, compared to the intact 25(OH)D(3). Methylamine was used as the mobile phase additive, and also effectively enhanced the assay sensitivity. Quantification was based on selected reaction monitoring, and 25-hydroxyvitamin D(4) was used as the internal standard. This method allowed the reproducible and accurate quantification of salivary 25(OH)D(3) using a 1.0-ml sample, and the limit of quantitation for 25(OH)D(3) was 2.0 pg/ml. The applicability of the developed method for clinical studies was then examined. There was a positive linear relationship (r (2) = 0.830) between the serum 25(OH)D(3) level, which is conventionally used as a means of assessing the vitamin D status, and the salivary 25(OH)D(3) level measured using the proposed method. The method also enabled the detection of the increase in the salivary 25(OH)D(3) level after the supplementation of vitamin D(3).

Studies on neurosteroids XXII. Liquid chromatography-tandem mass spectrometric method for profiling rat brain 3-oxo-4-ene-neuroactive steroids.

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the simultaneous determination of five 3-oxo-4-ene-neuroactive steroids, i.e. androstenedione, testosterone (T), progesterone (PROG), 20alpha-dihydroprogesterone and 20beta-dihydroprogesterone, in rat brain has been developed and validated. The brain steroids were extracted with methanol-acetic acid, purified using solid-phase extraction cartridges and subjected to LC-ESI-MS/MS. The method does not require derivatization. Deuterium-labeled T and PROG were used as the internal standards, and quantification was based on the selected reaction monitoring mode. This method allowed the reproducible and accurate quantification of the brain neuroactive steroids using 100 mg of tissue; the intra- and inter-assay relative standard deviations were below 4.7 and 4.3%, respectively, and the accuracy values were 97.6-103.2% for all the steroids. The limits of quantitation were 0.1 ng/g tissue for all the steroids. The application of this developed method for the analysis of changes in the brain neuroactive steroid levels by immobilization stress is also presented.

Studies on neurosteroids XXIII. Analysis of tetrahydrocorticosterone isomers in the brain of rats exposed to immobilization using LC-MS.

The identification and quantification of tetrahydrocorticosterone isomers (THBs; 3alpha,5alpha-, 3beta,5alpha-, 3alpha,5beta- and 3beta,5beta-THB) in rat brains using liquid chromatography (LC)-mass spectrometry (MS) are described. For the identification, the THBs were converted to the atmospheric pressure chemical ionization (APCI)-active derivatives, i.e., the dinitrobezoyl esters and 2-nitro-4-trifluoromethylphenyl hydrazones, and detected in the negative-ion mode. These derivatives showed 60- and 40-fold higher sensitivities, respectively, than intact steroids measured in the positive-APCI-MS. The derivatized THBs were satisfactorily separated from the others during the reversed-phase LC. The THBs were not detected at all in the brains of the unstressed rats. When the rats were exposed to the immobilization for 20 min, 3alpha,5alpha- and 3beta,5alpha-THB were detected as the major metabolites together with small amounts of 3alpha,5beta- and 3beta,5beta-THB in the male rat brain, while only 3alpha,5alpha-THB was detected in the female rats. Thus, the steroid variety found in the brains was different between the sexes. In the next step, 3alpha,5alpha-THB, a major metabolite found in the brains of the stressed rats, was quantified as its dinitrobezoyl ester. This method was accurate and reproducible, and the limit of quantitation was 1.0 ng/g tissue when a 50 mg tissue sample was used. There was also a sex difference in the brain 3alpha,5alpha-THB level; it was significantly higher in the female rats than in the male rats (P<0.05), although the brain corticosterone level was not higher in the stressed female rats than in the male rats (no statistical difference).

Determination of digoxin in human serum using stable isotope dilution liquid chromatography/electrospray ionization-tandem mass spectrometry.

A method for the determination of digoxin in human serum using a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) technique is reported. Digoxin and the internal standard, [21,21,22-(2)H(3)]digoxin, were extracted from 250 mul of human serum using a solid phase extraction cartridge (Oasis HLB) and analyzed by LC/ESI-MS/MS in the selected reaction monitoring mode. The intra- and inter-assay reproducibility and accuracy were satisfactory within the quantification range of 0.20-3.20 ng/ml. The concentrations of digoxin in the serum samples obtained from digitalized patients (n=19) were in the range of 0.25-2.84 ng/ml, which were compared to those obtained by radioimmunoassay.

Studies on Neurosteroids XXI: an improved liquid chromatography-tandem mass spectrometric method for determination of 5alpha-androstane-3alpha,17beta-diol in rat brains.

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the determination of the rat brain 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-Adiol) has been developed and validated. The brain extract was purified using solid-phase extraction cartridges, derivatized with isonicotinoyl azide, and subjected to LC-MS/MS. The method was accurate and reproducible, and the limit of quantitation was 0.1 ng/g tissue when a 100-mg tissue sample was used. The change in the brain 3alpha,5alpha-Adiol level by immobilization stress was also analyzed using the developed method.

Development and application of electrospray-active derivatization reagents for hydroxysteroids.

New derivatization reagents, 1-(2,4-dinitro-5-fluorophenyl)-4-methylpiperazine (PPZ) and 4-(4-methyl-1-piperazyl)-3-nitrobenzoyl azide (APZ), were developed for the liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) determination of steroids having a hydroxy group. PPZ reacted with a phenolic hydroxy group in estrogens. After quaternarization of the PPZ-estrogens with methyl iodide, the resulting derivatives provided more than a 2000-fold higher sensitivity compared to the intact estrogens. After derivatization of steroids having an alcoholic hydroxy group (5-ene-steroids or 5alpha-reduced steroids) with APZ followed by methylation, their detection responses increased more than 500 times. These derivatization procedures coupled with LC-ESI-MS/MS were successfully used for the determination of estrogens in the serum and prostatic 5alpha-dihydrotestosterone.

Alternative procedure for charged derivatization to enhance detection responses of steroids in electrospray ionization-MS.

A derivatization procedure has been examined to enhance the electrospray ionization (ESI)-MS detectabilities of steroids that charged derivatization is not suitable for. The derivatization procedure with 2-hydrazinopyridine or isonicotinoyl azide was very effective for the sensitive detection of di-oxosteroids or di-hydroxysteroids, respectively, and the detection limits of the resulting derivatives were as low as about 2 fmol. The derivatives also provided intense characteristic product ions in the MS-MS, which are expected to be usable for the selected reaction monitoring mode.