Determination of (13)C/(12)C ratios of endogenous urinary steroids excreted as sulpho conjugates

The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry‐based method for the measurement of stable carbon isotopes of endogenous urinary steroids excreted as sulphates is presented. The key element in sample preparation is the consecutive cleanup with high‐performance liquid chromatography of underivatized and acetylated steroids, which allows the isolation of seven analytes (pregn‐5‐ene‐3β,17α,20α‐triol, etiocholanolone, androsterone, epiandrosterone, dehydroepiandrosterone (DHEA), androst‐5‐ene‐3β,17β‐diol and androst‐5‐ene‐3β,17α‐diol) from a single urine specimen. These steroids are of particular importance to doping controls as they should enable the sensitive and retrospective detection of DHEA abuse by athletes. Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers the items, repeatability and reproducibility. The specificity was further demonstrated by gas chromatography/mass spectrometry for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring ¹³C/¹²C ratios and urinary concentrations of all implemented steroids, a reference population of n = 67 subjects was measured to enable the calculation of reference limits for all relevant steroidal Δ values. The applicability of the developed method was tested by means of a DHEA excretion study. Despite the fact that orally ingested DHEA is preferentially converted into sulphated metabolites and that the renal clearance of sulphated steroids is slow, only the ¹³C/¹²C ratio of EpiA demonstrated the potential to prolong the detection time for DHEA misuse. Copyright © 2010 John Wiley & Sons, Ltd.     

Gas chromatography flow rates for determining deuterium/hydrogen ratios of natural gas by gas chromatography/high-temperature conversion/isotope ratio mass spectrometry

The effects of the gas chromatography flow rate on the determination of the deuterium/hydrogen (D/H) ratios of natural gas utilising gas chromatography/high-temperature conversion/isotope ratio mass spectrometry (GC/TC/IRMS) have been evaluated. In general, the measured deltaD values of methane, ethane and propane decrease with increase in column flow rate. When the column flow rate is 1 mL/min or higher, which is commonly used for the determination of D/H ratios of natural gas, the organic H in gas compounds may not be completely converted into hydrogen gas. Based on the results of experiments conducted on a GC column with an i.d. of 0.32 mm, a GC flow rate of 0.6 mL/min is proposed for determining the D/H ratios of natural gas by GC/TC/IRMS. Although this value may be dependent on the instrument conditions used in this work, we believe that correct deltaD values of organic compounds with a few carbon atoms are obtained only when relatively low GC flow rates are used for D/H analysis by GC/TC/IRMS. Moreover, as the presence of trace water could significantly affect the determination of D/H ratios, a newly designed inlet liner was used to remove trace water contained in some gas samples.     

Determination of 13C/12C ratios of endogenous urinary steroids: method validation, reference population and application to doping control purposes

The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS)-based method for stable carbon isotopes of endogenous urinary steroids is presented. The key element in sample preparation is the consecutive cleanup with high-performance liquid chromatography (HPLC) of underivatized and acetylated steroids, which allows the isolation of ten analytes (11beta-hydroxyandrosterone, 5alpha-androst-16-en-3beta-ol, pregnanediol, androsterone, etiocholanolone, testosterone, epitestosterone, 5alpha-androstane-3alpha,17beta-diol, 5beta-androstane-3alpha,17beta-diol and dehydroepiandrosterone) from a single urine specimen. These steroids are of particular importance to doping controls as they enable the sensitive and retrospective detection of steroid abuse by athletes.Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers repeatability and reproducibility. Specificity was further demonstrated by gas chromatography/mass spectrometry (GC/MS) for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring (13)C/(12)C ratios of all implemented steroids, a reference population of n = 61 subjects was measured to enable the calculation of reference limits for all relevant steroidal Delta values.     

Hydropyrolysis over a platinum catalyst as a preparative technique for the compound‐specific carbon isotope ratio measurement of C27 steroids

Compound‐specific stable carbon isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is an important method for the determination of the ¹³C/¹²C ratios of biomolecules such as steroids, for a wide range of applications. However, steroids in their natural form exhibit poor chromatographic resolution, while derivatisation adds carbon thereby corrupting the stable isotopic composition. Hydropyrolysis with a sulphided molybdenum catalyst has been shown to defunctionalise the steroids, while leaving their carbon skeleton intact, allowing for the accurate measurement of carbon isotope ratios. The presence of double bonds in unsaturated steroids such as cholesterol resulted in significant rearrangement of the products, but replacing the original catalyst system with one of platinum results in higher conversions and far greater selectivity. The improved chromatographic performance of the products should allow GC/C/IRMS to be applied to more structurally complex steroid hormones and their metabolites. Copyright © 2010 John Wiley & Sons, Ltd.     

Metabolism of methyltestosterone in the greyhound

Gas chromatography/mass spectrometry and selective derivatisation techniques have been used to identify urinary metabolites of methyltestosterone following oral administration to the greyhound. Several metabolites were identified including reduced, mono‐, di‐ and trihydroxylated steroids. The major metabolites observed were 17α‐methyl‐5β‐androstane‐3α‐17β‐diol, 17α‐methyl‐5β‐androstane‐3α,16α,17β‐triol, and a further compound tentatively identified as 17α‐methyl‐5z‐androstane‐6z,17β‐triol. The most abundant of these was the 17α‐methyl‐5β‐androstane‐3α,16α,17β‐triol. This metabolite was identified by comparison with a reference standard synthesised using a Grignard procedure and characterised using trimethylsilyl (TMS) and acetonide‐TMS derivatisation techniques. There did not appear to be any evidence for 16β‐hydroxylation as a phase I metabolic transformation in the greyhound. However, significant quantities of 16α‐hydroxy metabolites were detected. Selective enzymatic hydrolysis procedures indicated that the major metabolites identified were excreted as glucuronic acid conjugates. Metabolic transformations observed in the greyhound have been compared with those of other mammalian species and are discussed here. Copyright © 2009 John Wiley & Sons, Ltd.     

Sterols from the Stems of Momordica charantia

A new sterol, 5α,6α‐epoxy‐3β‐hydroxy‐(22E,24R)‐ergosta‐8,22‐dien‐7‐one ( 1 ), together with eight known sterols, 5α,6α‐epoxy‐(22E,24R)‐ergosta‐8,22‐diene‐3β,7α‐diol ( 2 ), 5α,6α‐epoxy‐(22E,24R)‐ergosta‐8,22‐diene‐3β,7β‐diol ( 3 ), 5α,6α‐epoxy‐(22E,24R)‐ergosta‐8(14),22‐diene‐3β,7α‐diol ( 4 ), 3β‐hydroxy‐(22E,24R)‐ergosta‐5,8,22‐trien‐7‐one ( 5 ), ergosterol peroxide ( 6 ), clerosterol ( 7 ), decortinol ( 8 ), and decortinone ( 9 ), were isolated from the stems of Momordica charantia. Their structures were elucidated by mean of extensive spectroscopic methods, including ¹H, ¹³C, 2D‐NMR and HR‐EI‐MS, as well as comparison with the literature data. Compounds 1 , 4 , 5 , 8 , and 91 were not cytotoxic against the SK‐Hep 1 cell line.     

Analysis of [U‐13C6]glucose in human plasma using liquid chromatography/isotope ratio mass spectrometry compared with two other mass spectrometry techniques

The use of stable isotope labelled glucose provides insight into glucose metabolism. The ¹³C‐isotopic enrichment of glucose is usually measured by gas chromatography/mass spectrometry (GC/MS) or gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). However, in both techniques the samples must be derivatized prior to analysis, which makes sample preparation more labour‐intensive and increases the uncertainty of the measured isotopic composition. A novel method for the determination of isotopic enrichment of glucose in human plasma using liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) has been developed. Using this technique, for which hardly any sample preparation is needed, we showed that both the enrichment and the concentration could be measured with very high precision using only 20 µL of plasma. In addition, a comparison with GC/MS and GC/IRMS showed that the best performance was achieved with the LC/IRMS method making it the method of choice for the measurement of ¹³C‐isotopic enrichment in plasma samples. Copyright © 2009 John Wiley & Sons, Ltd.     

胆甾-4α-甲基-8-烯-3β，7α-二醇二乙酸酯的结构与性质研究

Lophenol, cholest-4α-methyl-7-en-3β-ol (1), obtained from Dracaena cochinchinensis (Lour.) S. C. Chen, was structurally modified. It was acetylated to protect 3β-hydroxyl group, and then oxidised by selenium dioxide in acetic acid to give cholest-4a-methyl-8-en-3β, Ta-diol diacetate (3). This compound 3 is unstable in chloroform solution or when heated and easily converted to a diene compound, cholest-4a-methyl-7,14-dien-3β-ol acetate (4). The structures of 3 and 4 were elucidated by means of IR, ^1H NMR, ^13C NMR and MS, and the absolute configuration of 3 was established by X-ray crystallography. The property of 3 was also discussed in this paper. Both 3 and 4 are new compounds and were reported for the first time.     

Simultaneous Determination 9 Anabolic Steroids Residues in Animal Muscle Tissues by Gas Chromatography‐Mass Spectrometry

Abstract

A method was developed for determining 9 anabolic steroids (ASs) residues in animal muscle tissues by gas chromatography‐mass spectrometry (GC/MS). After undergoing enzymolysis, being homogenized, and then being picked‐up by ultrasound the sample was extracted with tert‐Butyl methyl ether, cleaned up through solid‐phase extraction (SPE) based on the reverse phase (RP) principle, and after derivatization of the analyst of interest, analyzed by GC/MS under selective ion monitoring (SIM). The limits of the detection (LOD) of the GC/MS method used for testing epitestosterone (ETS), nandrolon (17β‐NT), 17α‐methyl‐testosterone (MTS), testosterone 17‐propionate (PTS), 17β‐estradiol 3‐benzoate (BES), estrone (ESN), 17β‐estradiol (17β‐ES), 17α‐ethynylestradiol (EES), and estriol (EST) in animal muscle ranged from 0.10 to 0.33 µg/kg and the limits of quantification (LOQ) were from 0.24 to 0.82 µg/kg. The experiments using spiked samples, such as pork, beef, chicken, and fish, made it clear that at addition level of 2.0 µg/kg, the average recovery of the ASs ranged from 62.5% to 80.5%, and the coefficient of variation ranged from 12.5% to 26.8%, while at an addition level of 5.0 µg/kg, the average recovery was from 70.8% to 86.4%, and the coefficient of variation was from 8.8% to 18.4%.     

Cytotoxic Activity of Capnellene‐8β, 10α‐Diol Derivatives from a Taiwanese Soft Coral Capnella sp.

The sesquiterpene capnellene‐8β, 10α‐diol ( 1 ) was isolated from non‐polar extract of the soft coral Capnella sp. Ten acylation products of capnellene‐8β, 10α‐diol were prepared: 10α‐hydroxy‐8β‐O‐benzoylcapnellene ( 2 ), 10α‐hydroxy‐8β‐O‐p‐toluoylcapnellene ( 3 ), 10α‐hydroxy‐8β‐O‐4‐chlorobenzoyl‐capnellene ( 4 ), 10α‐hydroxy‐8β‐O‐2‐furoylcapnellene ( 5 ), 10α‐hydroxy‐8β‐O‐2‐thiophenoylcapnellene ( 6 ), 10α‐hydroxy‐8β‐O‐4‐fluorobenzoylcapnellene ( 7 ), 10α‐hydroxy‐8β‐O‐4‐propylbenzoylcapnellene ( 8 ), 10α‐hydroxy‐8β‐O‐cinnamoylcapnellene ( 9 ), 10α‐hydroxy‐8β‐O‐4‐nitrobenzoylcapnellene ( 10 ), and 10α‐hydroxy‐8β‐O‐4‐anisoylcapnellene ( 11 ). The structures of capnellene‐8β, 10α‐diol as well as its derivatives were established through standard spectroscopic analysis. The in vitro cytotoxic activities of the eleven compounds were evaluated against Hela, KB, Daoy, and WiDr human tumor cell lines.     

1H and 13C NMR signal assignments of carboxyl‐linked glucosides of bile acids

Complete ¹H and ¹³C resonance assignments were carried out for a new type of carboxyl‐linked glucosides of chenodeoxycholic (3α,7α‐dihydroxy‐5β‐cholan‐24‐oic) and hyodeoxycholic (3α,6α‐dihydroxy‐5β‐cholan‐24‐oic) acids by using several homonuclear (¹H–¹H) and heteronuclear (¹H–¹³C) 2D NMR techniques. Differences in the ¹H and ¹³C resonances between the α‐ and β‐anomers of the ester glucosides of bile acids were clarified for the first time. A comparison of the ¹H and ¹³C signal shifts induced by β‐D ‐glucosidation at the 24‐carboxyl and 3α‐hydroxyl groups in the parent 5β‐cholanoic acid was also made. Copyright © 2003 John Wiley & Sons, Ltd.     

Comparison of different mass spectrometry techniques in the measurement of L‐[ring‐13C6]phenylalanine incorporation into mixed muscle proteins

Precise measurement of low enrichment of stable isotope labeled amino‐acid tracers in tissue samples is a prerequisite in measuring tissue protein synthesis rates. The challenge of this analysis is augmented when small sample size is a critical factor. Muscle samples from human participants following an 8 h intravenous infusion of L‐[ring‐¹³C₆]phenylalanine and a bolus dose of L‐[ring‐¹³C₆]phenylalanine in a mouse were utilized. Liquid chromatography tandem mass spectrometry (LC/MS/MS), gas chromatography (GC) MS/MS and GC/MS were compared to the GC‐combustion‐isotope ratio MS (GC/C/IRMS), to measure mixed muscle protein enrichment of [ring‐¹³C₆]phenylalanine enrichment. The sample isotope enrichment ranged from 0.0091 to 0.1312 molar percent excess. As compared with GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS showed coefficients of determination of R² = 0.9962 and R² = 0.9942, and 0.9217 respectively. However, the precision of measurements (coefficients of variation) for intra‐assay are 13.0%, 1.7%, 6.3% and 13.5% and for inter‐assay are 9.2%, 3.2%, 10.2% and 25% for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. The muscle sample sizes required to obtain these results were 8 µg, 0.8 µg, 3 µg and 3 µg for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. We conclude that LC/MS/MS is optimally suited for precise measurements of L‐[ring‐¹³C₆]phenylalanine tracer enrichment in low abundance and in small quantity samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Can gas chromatography combustion isotope ratio mass spectrometry be used to quantify organic compound abundance?

Quantifying the concentrations of organics such as phospholipid fatty acids (PLFAs) and n‐alkanes and measuring their corresponding ¹³ C/¹² C isotope ratios often involves two separate analyses; (1) quantification by gas chromatography flame ionisation detection (GC‐FID) or gas chromatography/mass spectrometry (GC/MS), and (2) ¹³ C‐isotope abundance analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC‐C‐IRMS). This requirement for two separate analyses has obvious disadvantages in terms of cost and time. However, there is a history of using the data output of isotope ratio mass spectrometers to quantify various components; including the N and C concentrations of solid materials and CO₂ concentrations in gaseous samples. Here we explore the possibility of quantifying n‐alkanes extracted from sheeps' faeces and fatty acid methyl esters (FAMEs) derivatised from PLFAs extracted from grassland soil, using GC‐C‐IRMS. The results were compared with those from GC‐FID analysis of the same extracts. For GC‐C‐IRMS the combined area of the masses for all the ions (m/z 44, 45 and 46) was collected, referred to as 'area all', while for the GC‐FID analysis the peak area data were collected. Following normalisation to a common value for added internal standards, the GC‐C‐IRMS 'area all' values and the GC‐FID peak area data were directly compared. Strong linear relationships were found for both n‐alkanes and FAMEs. For the n‐alkanes the relationships were 1:1 while, for the FAMEs, GC‐C‐IRMS overestimated the areas relative to the GC‐FID results. However, with suitable reference material 1:1 relationships were established. The output of a GC‐C‐IRMS system can form the basis for the quantification of certain organics including FAMEs and n‐alkanes. Copyright © 2011 John Wiley & Sons, Ltd.     

Strong anion‐exchange liquid chromatography coupled with isotope ratio mass spectrometry using a Liquiface interface

The introduction of liquid chromatography coupled with isotope ratio mass spectrometry (LC/IRMS) as an analytical tool for the measurement of isotope ratios in non‐volatile analytes has somewhat simplified the analytical cycle from sample collection to analysis mainly due to the avoidance of the extensive sample processing and derivatisation that were necessary for gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Here we test the performance of coupling strong anion exchange to IRMS using only the second commercially available interface; the Liquiface. The system was modified from installation specification to improve peak resolution in the interface and maintain peak separation from the column to the mass spectrometer. The system performance was assessed by the determination of sensitivity, accuracy and precision attained from carbohydrate separations. The system performed satisfactorily after modifications, resulting in maintenance of peak resolution from column to mass spectrometer. The sensitivity achieved suggested that ～150 ng carbon could be analysed with acceptable precision (<0.3‰). Accuracy was maintained in the interface as determined by correlation with offline techniques, resulting in regression coefficient of r² = 0.98 and a slope of 0.99. The average precision achieved for the separation of seven monosaccharides was 0.36‰. The integration of a carbonate removal device limited the effect of background carbon perturbations in the mass spectrometer associated with eluent gradients, and the coupling of strong anion‐exchange chromatography with IRMS was successfully achieved using the Liquiface. Copyright © 2010 John Wiley & Sons, Ltd.     

A comprehensive procedure based on gas chromatography–isotope ratio mass spectrometry following high performance liquid chromatography purification for the analysis of underivatized testosterone and its analogues in human urine

The confirmation by GC/C/IRMS of the exogenous origin of pseudo-endogenous steroids from human urine samples requires extracts of adequate purity. A strategy based on HPLC sample purification prior to the GC/C/IRMS analysis of human urinary endogenous androgens (i.e. testosterone, androsterone and/or androstenediols), is presented. A method without any additional derivatization step is proposed, allowing to simplify the urine pretreatment procedure, leading to extracts free of interferences permitting precise and accurate IRMS analysis, without the need of correcting the measured delta values for the contribution of the derivatizing agent. The HPLC extracts were adequately combined to both reduce the number of GC/C/IRMS runs and to have appropriate endogenous reference compounds (ERC; i.e. pregnanediol, 11-keto-etiocholanolone) on each GC–IRMS run. The purity of the extracts was assessed by their parallel analysis by gas chromatography coupled to mass spectrometry, with GC conditions identical to those of the GC/C/IRMS assay. The method has been validated according to ISO17025 requirements (within assay precision below 0.3 ‰ ¹³C delta units and between assay precision below 0.6 ‰ ¹³C delta units for most of the compounds investigated) fulfilling the World Anti-Doping Agency requirements.     

A simple rapid method to precisely determine 13C/12C ratios of plant methoxyl groups

Stable isotope ratios of individual plant components have become a valuable tool for the determination of the geographical origin and authenticity of foodstuff. A recently published method with considerable potential in this context is the measurement of the deuterium/hydrogen (D/H) isotope ratios of plant matter methoxyl groups. The method entailed cleavage of methyl ethers or esters with hydriodic acid (HI) to form gaseous methyl iodide (CH₃I) and then measurement of the δ²H value of this gas. Here, as a follow up to a previous study, we describe a method for the rapid and precise δ¹³C analysis of plant matter methoxyl groups using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Conditions for sample preparation were investigated for isotope discrimination effects, the GC conditions optimized, the reproducibility of the measurement of standards undertaken, and the precision of the method defined. The reproducibility of the δ¹³C value determined for a CH₃I standard on 20 consecutive measurements was found to be 0.17‰. The method was also tested on four methoxyl‐rich plant components: vanillin, lignin, wood and pectin. The analytical precision obtained, expressed as the average standard deviation, for these compounds was found to be better than 0.13‰. The described procedure which is simple and rapid, allowing preparation and analysis of a sample within 1 h, produces accurate and reproducible isotopic measurements. We suggest that this validated δ¹³C method when employed together with the recently published δ²H method for two‐dimensional stable isotope studies of organic matter containing methoxyl groups will be of considerable value, e.g. for proving the authenticity of foodstuff. Copyright © 2009 John Wiley & Sons, Ltd.     

Bulk derivatization and direct injection of human cerebrospinal fluid for trace‐level quantification of endogenous estrogens using trap‐and‐elute liquid chromatography with tandem mass spectrometry

Although there are existing methods for determining estrogen in human bodily fluids including blood plasma and serum, very little information is available regarding estrogen levels in human cerebrospinal fluid (CSF), which is critical to assess in studies of neuroprotective functions and diffusion of neuroprotective estrogens across the blood–brain barrier. To address this problem, a liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of four endogenous estrogens (estrone, 17α‐estradiol, 17β‐estradiol, and estriol) in human CSF was developed. An aliquot (300 μL) of human CSF was bulk derivatized using dansyl chloride in the sample and 10 μL was directly injected onto a restricted‐access media trap column for protein removal. No off‐line sample extraction or cleanup was needed. The limits of detection of estrone, 17α‐estradiol, 17β‐estradiol, and estriol were 17, 28, 13, and 30 pg/mL, respectively, which is in the parts‐per‐trillion regime. The method was then applied to human CSF collected from ischemic trauma patients. Endogenous estrogens were detected and quantified, demonstrating the effectiveness of this method.     

Measurements by gas chromatography/pyrolysis/mass spectrometry: fundamental conditions in (2)H/(1)H isotope ratio analysis

Gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/P/IRMS) is a relatively new method for on-line determination of (2)H/(1)H isotope ratios. The influence of different parameters on the (2)H/(1)H isotope ratios obtained in GC/P/IRMS has been thoroughly studied using several flavor compounds, such as 5-nonanone, linalool, menthol, linalyl acetate and 4-decanolide. The requirement of "conditioning" the pyrolysis reactor to obtain reliable delta(2)H(V-SMOW) values is discussed. Furthermore, the influence of the carrier gas flow of the gas chromatograph on the completeness of pyrolysis and subsequently on the delta(2)H(V-SMOW) values is investigated in detail. The linear range of the compounds investigated is determined. The results show that calibrating the GC/P/IRMS system with secondary standard substances is absolutely necessary in order to obtain reliable delta(2)H(V-SMOW) values. In view of interlaboratory comparability, validation procedures are recommended.     

Structural and Dynamic Basis of Human Cytochrome P450 7B1: A Survey of Substrate Selectivity and Major Active Site Access Channels

Cytochrome P450 (CYP) 7B1 is a steroid cytochrome P450 7α‐hydroxylase that has been linked directly with bile salt synthesis and hereditary spastic paraplegia type 5 (SPG5). The enzyme provides the primary metabolic route for neurosteroids dehydroepiandrosterone (DHEA), cholesterol derivatives 25‐hydroxycholesterol (25‐HOChol), and other steroids such as 5α‐androstane‐3β,17β‐diol (anediol), and 5α‐androstene‐3β,17β‐diol (enediol). A series of investigations including homology modeling, molecular dynamics (MD), and automatic docking, combined with the results of previous experimental site‐directed mutagenesis studies and access channels analysis, have identified the structural features relevant to the substrate selectivity of CYP7B1. The results clearly identify the dominant access channels and critical residues responsible for ligand binding. Both binding free energy analysis and total interaction energy analysis are consistent with the experimental conclusion that 25‐HOChol is the best substrate. According to 20 ns MD simulations, the Phe cluster residues that lie above the active site, particularly Phe489, are proposed to merge the active site with the adjacent channel to the surface and accommodate substrate binding in a reasonable orientation. The investigation of CYP7B1–substrate binding modes provides detailed insights into the poorly understood structural features of human CYP7B1 at the atomic level, and will be valuable information for drug development and protein engineering.