Rapid and sensitive high-performance liquid chromatographic method for simultaneous determination of retinol, α-tocopherol, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human plasma with photodiode-array ultraviolet detection

A new rapid and sensitive high-performance liquid chromatographic method using 0.5 ml of plasma has been developed for the simultaneous determination of retinol (vitamin A), α-tocopherol (vitamin E), 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃. The eluate was monitored with a photodiode-array detector with two fixed wavelengths (267 nm for vitamin D, 292 nm for α-tocopherol and retinol). For all compounds, including internal standards, the method provides extraction recoveries greater than 81%. Detection limits were equal to or lower than 1.5 μg/l for the 4 vitamins. Linearity of standards was excellent (r>0.999 in all cases). Intra-day and inter-day precision were generally acceptable; the intra-day-assay C.V. was 7.7 for all compounds and the inter-day-assay C.V. was <9.2% except for the lower concentrations of 25-hydroxyvitamin D₃, 25-hydroxyvitamin D₂ and α-tocopherol (10.8, 11.8 and 11.9, respectively). The important properties of the present method are its ease of use, its rapidity, since sample preparation was achieved in 15 min and all the compounds were eluted in less than 15 min, and its small sample volume required (=0.5 ml), which enables it to be used in pediatric practice.     

Determination of 25-hydroxyvitamin D3 in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A method for the determination of 25-hydroxyvitamin D₃, the major metabolite of vitamin D₃ in human plasma, using a non-radioactive internal standard and reversed-phase high-performance liquid chromatography with UV detection (265 nm) has been developed. The method was applied to the determination of the metabolite in plasma from healthy subjects (n=25) and from patients with chronic renal failure (n=12). 25-Hydroxyvitamin D₃ 3-sulfate, a major conjugated metabolite of 25-hyroxyvitamin D₃, was also determined and the correlation between the concentrations of these metabolites was examined. The study showed that almost equal amounts of both compounds were detected in the plasma of healthy subjects, however, in two subjects, the amount of sulfate in the free form was found to be about twice as high as normally detected. In contrast, the free form was predominant in the plasma of patients with chronic renal failure and the sulfate was not detected in four patients.     

Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status

Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D₃ and its metabolic product—25(OH)D₃ has not been studied. We investigated this relationship in two separate populations: the first, individuals from Hawaii who received significant sun exposure; the second, subjects from a lactation study who received up to 6400 IU vitamin D₃/day for 6 months.Results (1) the relationship between circulating vitamin D₃ and 25(OH)D in both groups was not linear, but appeared saturable and controlled; (2) optimal nutritional vitamin D status appeared to occur when molar ratios of circulating vitamin D₃ and 25(OH)D exceeded 0.3; at this point, the V_max of the 25-hydroxylase appeared to be achieved. This was achieved when circulating 25(OH)D exceeded 100 nmol.We hypothesize that as humans live today, the 25-hydroxylase operates well below its V_max because of chronic substrate deficiency, namely vitamin D₃. When humans are sun (or dietary) replete, the vitamin D endocrine system will function in a fashion as do these other steroid synthetic pathways, not limited by substrate. Thus, the relationship between circulating vitamin D and 25(OH)D may represent what “normal” vitamin D status should be.     

Characterization of astaxanthin esters in Haematococcus pluvialis by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

After first being analyzed by HPLC, 4 free carotenoids, 15 astaxanthin monoesters, 12 astaxanthin diesters, and 3 astacin monoesters in Haematococcus pluvialis were identified by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-(APCI)MS). Identification of each compound was based on the characteristic fragment ions of the positive ion mode, negative ion mode, and MS(2). Astaxanthin esters were identified based on the loss of one or two fatty acids. In a positive ion mode, astaxanthin monoesters had characteristic fragment ions at m/z 597 [M+H-fatty acid](+) and m/z 579 and 561 that resulted from a continuous loss of water. The relative intensity of m/z 579 in MS(2) amounted to more than 80% of that of the molecular ion. In astaxanthin diesters, the intensity of m/z 561 occasionally was equal to that of m/z 579, but in general the former, amounting to 50 to 60% or more of the molecular ion, was stronger than the latter, which decreased to 20 to 30% of the molecular ion. In addition, a set of compounds with maximum absorbance at 400 nm, detected by high-performance liquid chromatography-diode array detector (HPLC-DAD), had strong characteristic fragment ions at m/z 871 and 593 in the positive ion mode MS(2). They were presumed to be linolenic acid or an isomer of omega-6-gamma-linolenic acid esters of astacin.     

Simplified method for the determination of 25-hydroxy and 1α,25-dihydroxy metabolites of vitamins D2 and D3 in human plasma application to nutritional studies

A simplified method for the determination of 25-hydroxy and 1α,25-dihydroxy metabolites of vitamins D₂ and D₃ in human plasma was developed. Plasma samples were deproteinizated and applied to a Bond Elut C₁₈ OH cartridge to separate 25-hydroxyvitamin D (25-OH-D) and 1α-25-dihydroxyvitamin D [1,25(OH)₂D] fractions. The 25-OH-D fraction was purified by a Bond Elut C₁₈ cartridge and 25-OH-D₂ and 25-OH-D₃ were assayed by HPLC using a Zorbax SIL column. The 1,25(OH)₂D fraction obtained above was subsequently applied to HPLC using a Zorbax SIL column to separate 1,25(OH)₂D₂ and 1,25(OH)₂D₃ fractions which were determined by a radioreceptor assay (RRA) using calf thymus receptor. The method was applied to nutritional studies.     

Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status

Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D₃ and its metabolic product—25(OH)D₃ has not been studied. We investigated this relationship in two separate populations: the first, individuals from Hawaii who received significant sun exposure; the second, subjects from a lactation study who received up to 6400 IU vitamin D₃/day for 6 months.

Results (1) the relationship between circulating vitamin D₃ and 25(OH)D in both groups was not linear, but appeared saturable and controlled; (2) optimal nutritional vitamin D status appeared to occur when molar ratios of circulating vitamin D₃ and 25(OH)D exceeded 0.3; at this point, the V_max of the 25-hydroxylase appeared to be achieved. This was achieved when circulating 25(OH)D exceeded 100 nmol.

We hypothesize that as humans live today, the 25-hydroxylase operates well below its V_max because of chronic substrate deficiency, namely vitamin D₃. When humans are sun (or dietary) replete, the vitamin D endocrine system will function in a fashion as do these other steroid synthetic pathways, not limited by substrate. Thus, the relationship between circulating vitamin D and 25(OH)D may represent what “normal” vitamin D status should be.     

Synthesis of two carboxylic haptens for raising antibodies to 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3

Hapten derivatives of 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ were synthesized using the Wittig–Horner approach. Both haptens bearing a carboxylic group at the side chain that can be linked to a protein for raising antibodies of potential utility for the determination of 25-hydroxyvitamin D₃, 1α,25-dihydroxyvitamin D₃ and 1α-hydroxylated vitamin D₃ analogues.     

Pharmacokinetics of ergosterol in rats using rapid resolution liquid chromatography-atmospheric pressure chemical ionization multi-stage tandem mass spectrometry and rapid resolution liquid chromatography/tandem mass spectrometry

Rapid resolution liquid chromatography/tandem multi-stage mass spectrometry (RRLC-MS(n)) and rapid resolution liquid chromatography/tandem mass spectrometry (RRLC/MS/MS) methods were developed for the identification and quantification of ergosterol and its metabolites from rat plasma, urine and faeces. Two metabolites (ERG1 and ERG2) were identified by RRLC/MS(n). The concentrations of the ergosterol were determined by RRLC/MS/MS. The separation was performed on an Agilent Zorbax SB-C18 with the mobile phase consisting of methanol and water (containing 0.1% formic acid). The detection was carried out by means of atmospheric pressure chemical ionization mass spectrometry in positive ion mode with multiple reaction monitoring (MRM). Linear calibration curves were obtained in the concentration range of 7-2000, 6-2000 and 8-7500 ng/mL for plasma, urine and faecal homogenate, respectively. The intra- and inter-day precision values (RSD) were below 10%. The method was applied to the pharmacokinetic properties and elimination pathway of ergosterol in rats.     

Development of free 25-hydroxyvitamin D3 assay method using liquid chromatography-tandem mass spectrometry

The free hormone hypothesis has triggered controversies regarding the measurement of free vitamin D metabolites, such as free 25-hydroxyvitamin D (25(OH)D), as a suitable indicator for total vitamin D for clinical use. This issue can be addressed by developing a precise and accurate method for free 25(OH)D measurement. In the present study, a novel assay method for free 25(OH)D₃ based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Sample preparation first involved ultrafiltration to remove vitamin D-binding protein-bound and albumin-bound 25(OH)D, followed by extraction with a column, derivatization, evaporation, dissolution, and injection into the LC-MS/MS system. The coefficient of variation of repeatability and reproducibility obtained were 3.8–4.5% and 4.8–5.9%, respectively. Satisfactory linearity (r=0.999) was obtained up to 80 pg/ml. The lower quantification limit was 0.97 pg/ml and the S/N ratio on the peak of 1.0 pg/ml sample was 24.8 (which is more than the acceptable value of 10). The recovery rate was between 84.5 and 92.4% with a negligible matrix effect (94.5–104.9%). Levels of free 25(OH)D₃, but not total 25(OH)D₃, in the serum of the patients with chronic kidney disease (CKD) and hepatic cirrhosis (HC) were substantially lower than those in healthy subjects. The correlation coefficient between total and free 25(OH)D₃ was 0.738 in all samples, while the linear regression equations were different between the patients with CKD and HC. In conclusion, LC-MS/MS assay for free 25(OH)D₃ might be useful to evaluate high-throughput methods, including ELISA.     

Simple and rapid quantitative assay of C-labelled urea in human serum using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A simple and rapid quantitative method for ¹³C-labelled urea ([¹³C]urea) in human serum was developed by using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS). This method is used to establish and normalize the [¹³C]urea breath test, which is considered as an effective diagnostic method for Helicobacter pylori infection. HPLC-APCI-MS, involving a simple pretreatment process such as diluting serum with water, was shown to be able to discriminate the extrinsic [¹³C]urea from intrinsic urea present at high concentration in serum. In addition, a ¹³C nuclear magnetic resonance spectroscopic quantitative method for [¹³C]urea in human urine is also described. The precision and accuracy of measured concentrations in these two methods were found to be within the acceptable limit. An application of these methods to investigate the pharmacokinetic profile of orally administered [¹³C]urea in human serum and urine is also presented.     

Improved quantitative detection of 11 urinary phthalate metabolites in humans using liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry

Phthalates are widely used as industrial solvents and plasticizers, with global use exceeding four million tons per year. We improved our previously developed high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometric (HPLC-APCI-MS/MS) method to measure urinary phthalate metabolites by increasing the selectivity and the sensitivity by better resolving them from the solvent front, adding three more phthalate metabolites, monomethyl phthalate (mMP), mono-(2-ethyl-5-oxohexyl)phthalate (mEOHP) and mono-(2-ethyl-5-hydroxyhexyl)phthalate (mEHHP); increasing the sample throughput; and reducing the solvent usage. Furthermore, this improved method enabled us to analyze free un-conjugated mono-2-ethylhexyl phthalate (mEHP) by eliminating interferences derived from coelution of the glucuronide-bound, or conjugated form, of the mEHP on measurements of the free mEHP. This method for measuring phthalate metabolites in urine involves solid-phase extraction followed by reversed-phase HPLC-APCI-MS/MS using isotope dilution with (13)C(4) internal standards. We further evaluated the ruggedness and the reliability of the method by comparing measurements made by multiple analysts at different extraction settings on multiple instruments. We observed mMP, monoethyl phthalate (mEP), mono-n-butyl phthalate (mBP), monobenzyl phthalate (mBzP), mEHP, mEHHP and mEOHP in the majority of urine specimens analyzed with DEHP-metabolites mEHHP and mEOHP present in significantly higher amounts than mEHP.     

Determination of epimers 22R and 22S of budesonide in human plasma by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A highly sensitive and selective method has been developed for the simultaneous quantification of 22R- and 22S-epimers of budesonide in human plasma. The drug was isolated from human plasma using C₁₈ solid-phase extraction cartridges and was acetylated with a mixture of 12.5% acetic anhydride and 12.5% triethylamine in acetonitrile to form the 21-acetyl derivatives. Deuterium-labelled budesonide was synthesized and determined to have an isotopic purity > 99%. This was used as the internal standard. Epimers were quantified by automated liquid chromatography-atmospheric pressure chemical ionization mass spectrometry, operating in selected ion mode at m/z 473.2 and m/z 476.2. Linear responses were observed for both epimers over the range 0.25 to 10.0 ng/ml. The average recoveries of 22R- and 22S-epimers of budesonide from human plasma were 87.4% and 87.0%, respectively. The lower limit of quantification for each epimer was 0.25 ng/ml, corresponding to 50.0 pg of analyte on column. Within- and between-day coefficients of variation were 8.6% and 4.0%, respectively.     

Determination of testosterone concentrations in rat plasma using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry combined with ethyl oxime and acetyl ester derivatization

A quantitative method for measuring testosterone (T) concentrations in rat plasma was developed using ethyl oxime and acetyl ester derivatization and liquid chromatography-atmosphere pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS). The method utilizes a solid phase extraction with Varian Bond Elut C18, a derivatization process to form testosterone ethoxime acetate and LC-APCI-MS/MS with a reversed phase LC and a C8 column. This method is capable of detecting testosterone concentrations as low as 0.2 ng/ml in a 0.05 ml sample of rat plasma. This method can be used as a sensitive chromatography-based assay for small sample volumes of rat blood.     

Ternary solvent mixtures for improved resolution of hydroxylated metabolites of vitamin D2 and vitamin D3 during high-performance liquid chromatography

This paper reports the development of three new ternary solvent mixtures for the liquid-chromatographic separation of metabolites of vitamin D on microparticulate silica. All solvent systems offer reduced peak tailing and improved resolution of vitamin D compounds, particularly of 24(R),25-(OH)₂D₃, when compared to the commonly used hexane—isopropanol mixture. The new mixtures can be substituted for hexane—isopropanol systems presently used for preparative liquid-chromatographic steps prior to radioimmunoassay or competitive protein-binding assay of 24,25-(OH)₂D and 1,25-(OH)₂D in human plasma. Hexane—isopropanol—methanol (87:10:3) mixtures are recommended where the lipid content of samples is high, whereas hexane—ethanol—chloroform (80:10:10) promises to be a useful mixture for differentiating vitamin D₃ metabolites from their vitamin D₂ analogs. A combination of the two solvent systems permits the separate assay of both 24(R),25-(OH)₂D₃ and 24(R),25-(OH)₂D₂ as well as 1,25-(OH)₂D₃ and 1,25-(OH)₂D₂.     

Measurement and stability of FTY720 in human whole blood by high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug FTY720, using HPLC-tandem mass spectrometry. Whole blood samples (500 microl) were subjected to liquid-liquid extraction, in the presence of an internal standard (Y-32919). Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in positive ionization mode (FTY720: m/z 308.3-->255.3). The assay was linear from 0.2 to 25 microg/l (r(2)>0.997, n=5). The inter- and intra-day analytical recovery and imprecision for quality control samples (0.5, 7 and 15 microg/l) were 95.8-103.2 and <5.5%, respectively. At the lower limit of quantification (0.2 microg/l) the inter- and intra-day analytical recovery was 99.0-102.8% with imprecision of <7.6% (n=5). The assay had a mean relative recovery of 100.5+/-5.8% (n=15). Extracted samples were stable for 16 h. FTY720 quality control samples were stable at room temperature for 16 h, at 4 degrees C for at least 8 days and when taken through at least three freeze-thaw cycles. In conclusion, the method described displays analytical performance characteristics that are suitable for pharmacokinetic studies in humans.     

Vanadium and 1, 25 (OH)2 vitamin D3 combination in inhibitions of 1,2, dimethylhydrazine-induced rat colon carcinogenesis

The current investigation demonstrates the antitumor effects of combined supplementations of vanadium (V) (4.27 µmol/L drinking water ad libitum) and1α, 25-dihydroxy vitamin D₃ (Vitamin D₃) (0.3 μg/100 μL propylene glycol per os twice a week) on 1, 2 dimethylhydrazine (DMH) (20 mg/kg body weight) induced rat colon carcinogenesis. There was a significant reduction in incidence (70%), multiplicity (P < 0.0001) and volume (P < 0.01) of colon tumors. HPLC-fluorescence assay detected the combinatorial actions of V and Vitamin D₃ against DMH-induced colonic O⁶-methylguanine DNA adducts formation (at four sequential time points; ANOVA, F = 13.56, P < 0.01). Simultaneous inhibition of DNA single strand breaks (P < 0.001) indicates the potency of the combination regimen in limiting the initiation event of colon carcinogenesis. Immunohistochemical analysis revealed that the effect of V and vitamin D₃ occurred through suppression of cell proliferation (BrdU-LI: P < 0.001) along with an induction of apoptosis (TUNEL-LI: P < 0.01). The immunoexpression of tumor suppressor p53 and downregulation of antiapoptotic protein BCl-2 in subsequent immunofluorescence assay further provide strong evidence for the combinatorial inhibitory actions of vanadium and vitamin D₃ against DMH-induced rat colon carcinogenesis.     

Quantitative determination of cholesterol, sitosterol, and sitostanol in cultured Caco-2 cells by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

In this study, we describe a simple liquid extraction (methanol/choloroform, 1:1, v/v) method for endogenous free cholesterol and administered sterols extracted from cultured Caco-2 cells. To quantify sterol contents in Caco-2 cells, a new HPLC-APCI-MS method was developed. All the sterols were baseline separated using reversed-phase column (C8, 2.1 mm x 150 mm, 3.5 microm) and isocratic conditions (90%, v/v, methanol-water mixture containing 0.2 mM ammonium acetate). The full scan mass spectra of sterols were measured by an ion trap mass spectrometer equipped with an APCI ion source. The intense fragment ions resulting from the loss of water [M+H-H2O]+ (m/z 369, 395, 397 and 399 for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively) were used for determinations. The absolute extraction recovery of sterols from the spiked cell samples were 109.7+/-26.2, 105.7+/-5.1, 109.8+/-5.0 and 99.0+/-7.0% for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively. Furthermore, no significant matrix effect was observed for the sterols in the cell samples. The sample assay was based on the internal standard method using stigmasterol as an internal standard. The method was linear over the concentration ranges of 0.45-9.0 microM (cholesterol) and 0.225-7.2 microM (sitosterol and sitostanol). The within- and between-day precision was less than 7% and accuracy ranged from 93.51 to 101.77%. The lowest limit of quantitation (LLOQ) was 0.225 microM for sitosterol and sitostanol, and 0.45 microM for cholesterol. The accuracy range was 95-106% and precision was lower than 9% for all LLOQ values.     

Identification of astaxanthin diglucoside diesters from snow alga Chlamydomonas nivalis by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A method is described for the identification of astaxanthin glucoside esters from snow alga Chlamydomonas nivalis by means of liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI). The method is based on the use of preparative HPLC and subsequent identification of astaxanthin diglucoside diesters by microbore LC-MS/APCI. The combination of these two techniques was used to identify more than 100 molecular species. The astaxanthin diglucoside diester, i.e. (all-E)-[di-(6-O-oleoyl-beta-D-glucopyranosyloxy)]-astaxanthin, was also synthesized to unambiguously confirm its structure.     

Permeabilization induced by lipid II-targeting lantibiotic nisin and its effect on the bioconversion of vitamin D3 to 25-hydroxyvitamin D3 by Rhodococcus erythropolis

Vitamin D₃ (VD₃) is a fat-soluble prohormone in mammals. VD₃ is inert and must be activated by hydroxylation at the C-25 and C-1α positions to exert its biological activity. We recently accomplished the bioconversion of VD₃ to 25(OH)VD₃ with a recombinant strain of Rhodococcus erythropolis and found that the permeability of VD₃ into the cytoplasm may be the rate-limiting step of 25(OH)VD₃ production (Sallam et al., 2010). When the cells were treated with the lipid II-targeting lantibiotic nisin, the permeability of green chemiluminescent cyclodextrin (GCCD), which is used as a model substrate instead of VD₃-partially methylated-β-cyclodextrin (PMCD) complex, was drastically induced. Nisin also induced VD₃ hydroxylation, and the rate was correlated with the expression levels of Vdh and its redox partner proteins. In the bioconversion reaction, the stability of the redox partner proteins and the additional NADH-regenerating system are crucial for VD₃ hydroxylation. The degradation rate of the [2Fe–2S] cluster of ferredoxin ThcC from R. erythropolis NI86/21 is faster than that of AciB from Acinetobacter sp. OC4. Therefore, the nisin-treated R. erythropolis cells coexpressing Vdh and AciBC (1176.5 μg) exhibited much greater 25(OH)VD₃ production than the cells coexpressing Vdh and ThcCD (431.7 μg) after four consecutive 16 h reactions. These results suggest that nisin forms nisin-lipid II pore complexes in the Rhodococcus membrane that increase the accessibility of VD₃–PMCD complexes to the inside of the cells. Furthermore, nisin-treated Rhodococcus cells can be utilized for the bioconversion of other fat-soluble chemicals.     

Evaluation of electrospray ionization and atmospheric pressure chemical ionization for simultaneous detection of estrone and its metabolites using high-performance liquid chromatography/tandem mass spectrometry

The levels of estrogens and/or their metabolites play important roles in carcinogenesis, reproductive function, and sexual development during perinatal and adolescence periods. The main purpose of this report was to investigate the applicability of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with electrospray ionization (ESI) and/or atmospheric pressure chemical ionization (APCI) for simultaneous detection of estrone (E1) and its six metabolites. Both positive and negative ionization modes in ESI and APCI were used to evaluate the signal responses of seven target analytes. Among the seven target analytes, five analytes, E1, 16alpha-hydroxyestrone, 2-methoxyestrone, 4-methoxyestrone, and 2-hydroxyestrone-3-methyl, produced signals with the best signal-to-noise (S/N) ratios in positive APCI-MS/MS mode, while the other two analytes, 2-hydroxyestrone and 4-hydroxyestrone, yielded the best S/N ratios in negative ESI-MS/MS mode. Based on the results of the evaluation, HPLC-APCI-MS/MS with switching between positive and negative modes was recommended for simultaneous detection of E1 and its six metabolites. The proposed analytical scheme was successfully applied in the analysis of cell culture medium of Human liver carcinoma cells treated with varying amounts of 2,3,7,8-tetrachlorodibenzo-p-dioxin.