Tatsuya Higashi

Gas chromatography and high-performance liquid chromatography of natural steroids

This review article underlines the importance of gas chromatography (GC), high-performance liquid chromatography (HPLC) and their hyphenated techniques using mass spectrometry (MS) for the determination of natural steroids, especially in human biological fluids. Steroids are divided into eight categories based on their structures and functions, and recent references using the above methodologies for the analysis of these steroids are cited. GC and GC-MS are commonly used for the determination of volatile steroids. Although HPLC is a widely used analytical method for the determination of steroids including the conjugated type in biological fluids, LC-MS is considered to be the most promising one for this purpose because of its sensitivity, specificity and versatility.

Studies on neurosteroids XVII. Analysis of stress-induced changes in neurosteroid levels in rat brains using liquid chromatography-electron capture atmospheric pressure chemical ionization-mass spectrometry.

The analysis of stress-induced changes in the brain neurosteroid levels by liquid chromatography (LC)-electron capture atmospheric pressure chemical ionization-mass spectrometry (ECAPCI-MS) is described. In the present method, neurosteroids were derivatized with a highly electron-affinitive reagent, 2-nitro-4-trifluoromethylphenylhydrazine (NFPH), to convert them to the corresponding hydrazones. The derivatized steroids showed over a 20-fold higher sensitivity in ECAPCI-MS than intact steroids measured by positive atmospheric pressure chemical ionization (APCI)-MS. Application of this method to the analysis of rat brain samples confirmed the significant increase in the levels of pregnenolone (PREG), progesterone (PROG), 5alpha-dihydroprogesterone (DHPROG), allopregnanolone (3alpha-hydroxy-5alpha-pregn-20-one; AP), and epiallopregnanolone (3beta-hydroxy-5alpha-pregn-20-one; EpiAP) in the fixated rats. The din stress, which we examined as a new short-term mental stress model, also elevated the brain neurosteroid levels. It is known that various types of stress lower the gamma-aminobutyric acid type A (GABA(A)) receptor function and induce the neuronal overexcitation. The increase in the brain level of AP, a potent positive modulator of GABA(A) receptors, may be the defensive response against acute stress. The increase in the brain concentration of its precursors, PREG, PROG, and DHPROG, may be associated with the acceleration of the AP synthesis. Thus, the present studies suggest that changes in the brain levels of neurosteroids may play an important role in the homeostatic mechanisms that counteract the inhibitory effect of stress on the GABA(A) receptor function.

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.

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.

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).

Simultaneous determination of 17α-hydroxypregnenolone and 17α-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.

Highly sensitive and positively charged precolumn derivatization reagent for amines and amino acids in liquid chromatography/electrospray ionization tandem mass spectrometry

We have developed a highly sensitive and positively charged precolumn derivatization reagent, (5-N-succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP), for amines and amino acids in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). The handling of the derivatization reaction is quite simple and the reagent reacts with the analytes rapidly and with high efficiency. The derivatized analytes were observed to form regular and intense product ions upon MS/MS analysis; thus, highly sensitive and selective detection was possible in the selected reaction monitoring (SRM) mode. The limits of detection of the SPTPP-derivatized analytes were less than sub-femtomole levels. The sensitivities of the derivatized analytes increased about 500-fold compared to those of underivatized analytes. Since the hydrophobicities of the samples increased after their derivatization, the resolution of the analytes improved dramatically when a reversed-phase system was used. The relative standard deviations of intra-day and inter-day variations were below 10.6% and 13.3%, respectively. The accuracy ranged between 86.6-113% and 83.4-113%, respectively. Furthermore, the developed reagent was used for the analysis of the neurotransmitter 4-aminobutanoic acid (GABA) and oxidative stress markers such as oxidized, nitrated, and halogenated tyrosines in rat serum.

Biomarker discovery in biological specimens (plasma, hair, liver and kidney) of diabetic mice based upon metabolite profiling using ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry

BACKGROUND The number of diabetic patients has recently been increasing worldwide. Diabetes is a multifactorial disorder based on environmental factors and genetic background. In many cases, diabetes is asymptomatic for a long period and the patient is not aware of the disease. Therefore, the potential biomarker(s), leading to the early detection and/or prevention of diabetes mellitus, are strongly required. However, the diagnosis of the prediabetic state in humans is a very difficult issue, because the lifestyle is variable in each person. Although the development of a diagnosis method in humans is the goal of our research, the extraction and structural identification of biomarker candidates in several biological specimens (i.e., plasma, hair, liver and kidney) of ddY strain mice, which undergo naturally occurring diabetes along with aging, were carried out based upon a metabolite profiling study. METHODS The low-molecular-mass compounds including metabolites in the biological specimens of diabetic mice (ddY-H) and normal mice (ddY-L) were globally separated by ultra-performance liquid chromatography (UPLC) using different reversed-phase columns (i.e., T3-C18 and HS-F5) and detected by electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The biomarker candidates related to diabetes mellitus were extracted from a multivariate statistical analysis, such as an orthogonal partial least-squares-discriminant analysis (OPLS-DA), followed by a database search, such as ChemSpider, KEGG and HMDB. RESULTS Many metabolites and unknown compounds in each biological specimen were detected as the biomarker candidates related to diabetic mellitus. Among them, the elucidation of the chemical structures of several possible metabolites, including more than two biological specimens, was carried out along with the comparison of the tandem MS/MS analyses using authentic compounds. One metabolite was clearly identified as N-acetyl-L-leucine based upon the MS/MS spectra and the retention time on the chromatograms. CONCLUSIONS N-acetyl-L-leucine is an endogenous compound included in all biological specimens (plasma, hair, liver and kidney). Therefore, this metabolite appears to be a potential biomarker candidate related to diabetes. Although the structures of other biomarker candidates have still not yet determined, the present approach based upon a metabolite profiling study using UPLC-ESI-TOF-MS could be helpful for understanding the abnormal state of various diseases.

Studies on neurosteroids XV. Development of enzyme-linked immunosorbent assay for examining whether pregnenolone sulfate is a veritable neurosteroid.

An enzyme-linked immunosorbent assay (ELISA) of pregnenolone sulfate (PREGS) has been developed for examining whether it is a veritable neurosteroid. 11alpha-Hemiglutaryloxy-PREGS was newly synthesized and conjugated with bovine serum albumin (BSA), which was injected to rabbits for the production of anti-PREGS antibodies. A bridge-heterologous ELISA system employing the sequential saturation method exhibited a high sensitivity with a midpoint of 30 pg. Although the antibody showed some cross-reactivity with PREG (4.4%), it easily discriminated other related steroids reported to exist in the mammalian brain. The rat brain homogenate was treated with hexane and subjected to an OASIS HLB cartridge, which was washed with AcOEt to remove the unconjugated steroids, and then the desired sulfate was eluted with EtOH. The recovery rate of PREGS through the pretreatment was satisfactory, but its brain levels in the preliminary experiments were much lower than those previously measured by gas chromatography (GC)-mass spectrometry (MS) after solvolysis. These results practically agreed with our previous results by liquid chromatography (LC)-electrospray ionization (ESI)-MS without deconjugation.

Characterization of urinary metabolites of vitamin D3 in man under physiological conditions using liquid chromatography-tandem mass spectrometry

The characterization of the urinary metabolites of vitamin D(3) in man under physiological conditions was performed using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The urine specimens obtained from healthy volunteers were treated with beta-glucuronidase, purified with disposal solid-phase extraction cartridges, derivatized with a Cookson-type reagent, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione, and subjected to LC-MS-MS. The derivatization was employed to increase the ionization efficiencies of the vitamin D(3) metabolites, which enabled detection of the metabolites in the picogram range. The identification of the genin parts of the metabolites was done by comparison with authentic samples based on their LC-MS-MS data. The glucuronides of 23S,25-dihydroxyvitamin D(3) and 24R,25-dihydroxyvitamin D(3) were obtained as the main metabolites from the urine in almost equal amounts. In contrast to the fact that the plasma/serum concentration of the former is much lower than that of the latter, the hydroxylation at the C-23 position was considered to be the important side-chain modification of 25(OH)D(3) to excrete the excess vitamin D(3) in man. In addition, 23S,25-dihydroxy-24-oxovitamin D(3) occurred as its glucuronide in most of the urine, which suggested that this metabolite also plays a part in the excretion of vitamin D(3) in man.