Determination of campesterol, stigmasterol, and beta-sitosterol in saw palmetto raw materials and dietary supplements by gas chromatography: single-laboratory validation

In conjunction with an AOAC Presidential Task Force on Dietary Supplements, a method was validated for measurement of 3 plant sterols (phytosterols) in saw palmetto raw materials, extracts, and dietary supplements. AOAC Official Method 994.10, "Cholesterol in Foods," was modified for purposes of this validation. Test samples were saponified at high temperature with ethanolic potassium hydroxide solution. The unsaponifiable fraction containing phytosterols (campesterol, stigmasterol, and beta-sitosterol) was extracted with toluene. Phytosterols were derivatized to trimethylsilyl ethers and then quantified by gas chromatography with a hydrogen flame ionization detector. The presence of the phytosterols was detected at concentrations greater than or equal to 1.00 mg/100 g based on 2-3 g of sample. The standard curve range for this assay was 0.00250 to 0.200 mg/mL. The calibration curves for all phytosterols had correlation coefficients greater than or equal to 0.995. Precision studies produced relative standard deviation values of 1.52 to 7.27% for campesterol, 1.62 to 6.48% for stigmasterol, and 1.39 to 10.5% for beta-sitosterol. Recoveries for samples fortified at 100% of the inherent values averaged 98.5 to 105% for campesterol, 95.0 to 108% for stigmasterol, and 85.0 to 103% for beta-sitosterol.     

Synthesis, isolation and characterisation of beta-sitosterol and beta-sitosterol oxide derivatives

Beta-sitosterol is the most prevalent plant cholesterol derivative (phytosterol) and can undergo similar oxidation to cholesterol, leading to beta-sitosterol oxides. The biological impact of phytosterol oxides has only been evaluated in a phytosterol blend (usually of beta-sitosterol, campesterol, stigmasterol and dihydrobrassicasterol). The lack of pure phytosterols, including beta-sitosterol, hinders the collection of significant toxicity data on the individual beta-sitosterol oxides. An efficient synthetic route to multi-gram quantities of pure beta-sitosterol is described here, together with the first syntheses and characterisation of pure beta-sitosterol oxides.     

Identification of beta-sitosterol, stigmasterol and ergosterin in A. roxburghii using supercritical fluid extraction followed by liquid chromatography/atmospheric pressure chemical ionization ion trap mass spectrometry

beta-Sitosterol and stigmasterol are the most common phytosterols in traditional Chinese medicine. They have been proved to have many important bioactivities. To the best of our knowledge, this is the first report that beta-sitosterol, stigmasterol and ergosterol coexisting in A. roxburghii herbs can be simultaneously extracted by a supercritical fluid extraction (SFE) procedure; then a simple high-performance liquid chromatography/atmospheric pressure chemical ionization ion trap mass spectrometry (HPLC/APCI/MS) method was developed for simultaneous identification and determination of these three compounds. The ion trap MS/MS detector was equipped with an atmospheric pressure chemical ionization source operating in the positive ion mode, APCI(+). The linear responses were obtained in the concentration range of 0.50-150 microg/mL (r = 0.9999) for ergosterol, 5-400 microg/mL (r = 0.9999) for stigmasterol, and 10-2000 microg/mL (r = 0.9998) for beta-sitosterol. An orthogonal L(9) (3(3)) test design was employed for optimization of the SFE process. Under the optimized conditions, i.e. pressure of 25 mPa, temperature of 45 degrees C and ethanol as modifier, the concentrations of sterols in the extract were found to be 2.89% (g/g) for beta-sitosterol, 3.56% (g/g) for stigmasterol and 2.96% (g/g) for ergosterin. The SFE method was also compared with a previously developed Soxhlet extraction. The SFE method produced higher yields of sterols than that of the Soxhlet extraction. The proposed method has been successfully used for identification and quantitation of beta-sitosterol, stigmasterol and ergosterin in a real A. roxburghii sample.     

Liquid chromatography-UV determination and liquid chromatography-atmospheric pressure chemical ionization mass spectrometric characterization of sitosterol and stigmasterol in soybean oil.

A narrow-bore HPLC-UV method was developed for the analysis of two of the more abundant naturally occurring phytosterols in vegetable oils: sitosterol and stigmasterol. The method enabled detection of the compounds at a concentration of 0.42 microg/ml and quantitation at concentrations of 0.52 and 0.54 microg/ml for sitosterol and stigmasterol, respectively. An excellent linearity was determined over two orders of concentration magnitude (r2 0.999-1.000) and verified by applying the Mandel fitting test (p>0.099) and the lack-of-fit test (p>0.057) performed at the 95% confidence level. A good intra-day precision ranging from 0.15 to 1.16% was calculated at two concentration levels (2 and 100 microg/ml). The inter-day reproducibility was verified on 3 different days by performing an homoscedasticity test and analysis of variance. A solid-phase extraction method was developed on silica cartridges for the isolation of phytosterols from soybean oil providing recovery values of 101+/-9 and 106+/-7% for sitosterol and stigmasterol, respectively. Good accuracy of the method was statistically demonstrated since no matrix effect was found for both the analytes. The developed method was applied to the quantitative assay of phytosterols in a soybean oil sample (61+/-5 mg/100 g of stigmasterol and 118+/-4 mg/100 g sitosterol). The HPLC-atmospheric pressure chemical ionization MS technique enabled the identification of stigmasterol, sitosterol and campesterol in the oil sample.     

Gas chromatographic properties of common cholesterol and phytosterol oxidation products

The most common cholesterol and phytosterol oxidation products found in foodstuffs or biological matrices are the 7alpha- and 7beta-hydroxysterol, 7-ketosterol, 5alpha,6alpha- and 5beta,6beta-epoxysterol, and triol derivatives of sterols. This study focused on the preparation and purification of such products derived from campesterol, stigmasterol and beta-sitosterol. The identity of the substances was confirmed by mass spectroscopic analysis. The elution order of a complex mixture composed of the 7alpha- and 7beta-hydroxysterol, 7-ketosterol, 5alpha,6alpha- and 5beta,6beta-epoxysterol, and triol derivatives of cholesterol, campesterol, stigmasterol and beta-sitosterol was recorded on an apolar as well as a medium-polarity capillary column in relation to two commonly used internal standards, i.e. alpha-cholestane and 19-hydroxy cholesterol. Flame-ionization detector as well as mass spectrometry response factors were derived from a gravimetrically prepared mixture of commercially available cholesterol oxide standards. It was proven that the ionization efficiency of cholesterol and phytosterol oxides are very similar and that response factors obtained for cholesterol oxidation products are also valid for quantitative work regarding phytosterol oxidation products.     

烟草中植物甾醇的GC-MS分析

建立了一种GC-MS同时测定烟草中胆甾醇、菜油甾醇、豆甾醇及β-谷甾醇的分析方法。结果表明:胆甾醇、菜油甾醇、豆甾醇及β-谷甾醇回收率在95.94～102.41%之间,RSD<6.01%。在所测定的烟草样品中,豆甾醇的含量较高。所检测的烟草样品中,均未检测出糖苷态胆甾醇。     

Extraction optimization by response surface methodology: Purification and characterization of phytosterol from sugarcane (Saccharum officinarum L.) rind

A green, simple, and effective method for the extraction of sugarcane lipids from sugarcane rind was investigated by response surface methodology. The optimum conditions of technological progress obtained through response surface methodology were as follows: liquid‐to‐solid ratio 7.94: 1 mL/g, extraction temperature 50°C and extraction time 5.98 h. The practical sugarcane lipids extraction yield was 6.55 ± 0.28%, which was in good consistence with the predicted extraction yield of 6.47%. The results showed that the sugarcane lipids extraction yield obtained in optimum conditions increased by 1.16～7.28‐fold compared to the yields obtained in single‐factor experiments. After saponification and SPE steps, the nonsaponifiable fraction of sugarcane lipids was analyzed by gas chromatography with mass spectrometry and high‐performance liquid chromatography. β‐Sitosterol, stigmasterol, and campesterol were the prevailing phytosterols in the sample, while fucosterol, gramisterol, stigmast‐7‐en‐3‐ol, (3β,5α,24S)‐, stigmasta‐4,6,22‐trien‐3α‐ol, and cholest‐8(14)‐en‐3β‐ol acetate were also identified as minor steroids. Furthermore, the content of β‐sitosterol and a mixture of campesterol and stigmasterol (quantified by high‐performance liquid chromatography) was 44.18 mg/100 g dry weight and 43.20 mg stigmasterol/100 g dry weight, respectively. Our results indicate that sugarcane rind is a good source of phytosterol.     

Development of liquid chromatographic methods for the determination of phytosterols in Standard Reference Materials containing saw palmetto

Liquid chromatographic (LC) methods using atmospheric pressure chemical ionization/mass spectrometric (APCI-MS) detection were developed for the separation and analysis of the phytosterols campesterol, cycloartenol, lupenone, lupeol, beta-sitosterol, and stigmasterol. Brassicasterol and cholesterol were also included for investigation as internal standards. The methods were used to identify and quantify the phytosterols in each of two Serenoa repens (saw palmetto) Standard Reference Materials (SRMs) developed by the National Institute of Standards and Technology (NIST). Values obtained by LC-MS were compared to those obtained using the more traditional approach of gas chromatography with flame ionization detection. This is the first reported use of LC-MS to determine phytosterols in saw palmetto dietary supplement materials.     

Single-laboratory validation of a method for the determination of furan in foods by using static headspace sampling and gas chromatography/mass spectrometry

A headspace gas chromatography/mass spectrometry method was developed and validated in-house for the determination of furan in foods. The method of standard additions with d4-furan as the internal standard was used to quantitate furan. The limit of detection and limit of quantitation (LOQ) values ranged from 0.2 and 0.6 nglg, respectively, in apple juice to 0.9 and 2.9 ng/g, respectively, in peanut butter. Recoveries were obtained at 0.5, 1, 2, and 3 times the LOQ. At 1, 2, and 3 times the LOQ, the recoveries ranged from 89.4 to 108%, and the relative standard deviations ranged from 3.3 to 17.3% for all the matrixes. For apple juice, chicken broth, and infant formula, the averaged coefficients of determination from the linear regression analyses were >0.99 with each food fortified at 0.5, 1, 2, and 3 times the LOQ. The coefficients of determination were >0.99 for green beans and 0.96 for peanut butter with the foods fortified at 1, 2, and 3 times the LOQ. Within-laboratory precision was determined by comparing the amounts of furan found in 18 samples by 2 analysts on different days with different instruments. For most of the foods, the difference between the amounts found by each analyst was <18%. The method was used to conduct a survey of >300 foods. The furan levels found ranged from none detected to 174 ng/g.     

Pressurized liquid extraction using water/isopropanol coupled with solid-phase extraction cleanup for industrial and anthropogenic waste-indicator compounds in sediment

A broad range of organic compounds is recognized as environmentally relevant for their potential adverse effects on human and ecosystem health. This method was developed to better determine the distribution of 61 compounds that are typically associated with industrial and household waste as well as some that are toxic and known (or suspected) for endocrine-disrupting potential extracted from environmental sediment samples. Pressurized liquid extraction (PLE) coupled with solid-phase extraction (SPE) was used to reduce sample preparation time, reduce solvent consumption to one-fifth of that required using dichloromethane-based Soxhlet extraction, and to minimize background interferences for full scan GC/MS analysis. Recoveries from spiked Ottawa sand, commercially available topsoil, and environmental stream sediment, fortified at 4-720 μg per compound, averaged 76 ± 13%. Initial method detection limits for single-component compounds ranged from 12.5 to 520 μg/kg, based on 25 g samples. Results from 103 environmental sediment samples show that 36 out of 61 compounds (59%) were detected in at least one sample with concentrations ranging from 20 to 100,000 μg/kg. The most frequently detected compound, beta-sitosterol, a plant sterol, was detected in 87 of the 103 (84.5%) environmental samples with a concentration range 360-100,000 μg/kg. Results for a standard reference material using dichloromethane Soxhlet-based extraction are also compared.     

Chemical composition and antioxidant,antibacterial activity of Cirsium rivulare (Jacq) All. roots

The mixture of three phytosterols (campesterol, stigmasterol and β-sitosterol), β-sitosterol 3-O-glucoside and syringin were isolated from hexane and methanol extract of Cirsium rivulare roots after chromatographic separation. The main component of the source was syringin which was obtained with the yield 0.08% of the dry source. In hexane extract, the qualitative and quantitative composition of fatty acids was determined. The predominant component was linoleic acid (23.31 mg/g of extract). The extracts showed antioxidant activity. The ability to scavenge DPPH? free radical was in correlation with appointed total phenol content. The not-defatted methanolic extract was the most active. Hexane and defatted methanol extracts showed moderate antibacterial activity against G(+) and G(?) strains with MIC and MBC ranged from 25 to 200 μg/mL.     

Simultaneous analysis of select pharmaceuticals and personal care products in fish tissue using pressurized liquid extraction combined with silica gel cleanup

Analytical improvements were developed and validated for measuring select personal care products (PCPs) and two pharmaceuticals in fish tissue. The method was validated using fortified fillet tissue for twelve PCPs including fragrance materials, alkylphenols, photo initiators, and triclosan as well as two pharmaceuticals including carbamazepine (anti-seizure) and diazepam (anti-convulsant). The analytical method utilized pressurized liquid extraction (PLE) combined with silica gel cleanup, gel permeation chromatography, and gas chromatography ion-trap tandem mass spectrometry. Silica gel cleanup was combined with the PLE to produce one automated extraction/cleanup technique. This analytical improvement served to reduce the incurred cost, time, and loss of potential target analytes associated with independent cleanup steps. The combined extraction/cleanup technique resulted in an average increase of 10% in analyte recoveries. Average triplicate recoveries and relative standard deviations for the entire method, using 2.5 g of fish fillet tissue, were 92 ± 9% (recoveries ranged from 64 to 131%). The sensitivity of the analytical methods was improved by optimizing the resonant collision induced dissociation energy to the hundredths place (0.01 V). Improvements in ion production range from 24 to 122% for six of the 12 PCPs. Statistically derived method detection limits (MDLs) were also lowered on average by a factor of 8 and ranged from 1.2 to 38 ng/g wet weight. MDLs for carbamazepine and diazepam were 18 and 3.7 ng/g wet weight, respectively. Galaxolide and tonalide were measured in an environmental sample at concentrations of 81 and 5.5 ng/g wet weight, respectively.     

MSPD Procedure for Determination of Carbofuran, Pyrimethanil and Tetraconazole Residues in Banana by GC–MS

An extraction method based on matrix solid-phase dispersion was developed to determine carbofuran, pyrimethanil and tetraconazole in banana using gas chromatography–mass spectrometry. The best results were obtained using 2.0 g of banana, 1.0 g of silica as dispersant sorbent and n-hexane:ethyl acetate (1:4, v/v) as eluting solvent. The method was validated using banana samples fortified with pesticides at different concentration levels (0.05–2.0 mg kg^?1). Average recoveries (four replicates) ranged from 68 to 111%, with relative standard deviations between 6.6 and 20.5%. Detection and quantification limits for banana ranged from 0.02 to 0.05 and 0.05 to 0.10 mg kg^?1, respectively.     

Determination of submicrogram-per-liter concentrations of caffeine in surface water and groundwater samples by solid-phase extraction and liquid chromatography

A method for determining submicrogram-per-liter concentrations of caffeine in surface water and groundwater samples has been developed. Caffeine is extracted from a 1 L water sample with a 0.5 g graphitized carbon-based solid-phase cartridge, eluted with methylene chloride-methanol (80 + 20, v/v), and analyzed by liquid chromatography with photodiode-array detection. The single-operator method detection limit for organic-free water samples was 0.02 microgram/L. Mean recoveries and relative standard deviations were 93 +/- 13% for organic-free water samples fortified at 0.04 microgram/L and 84 +/- 4% for laboratory reagent spikes fortified at 0.5 microgram/L. Environmental concentrations of caffeine ranged from 0.003 to 1.44 micrograms/L in surface water samples and from 0.01 to 0.08 microgram/L in groundwater samples.     

Determination of amphetamine-type stimulants, cocaine and ketamine in human hair by liquid chromatography/linear ion trap-Orbitrap hybrid mass spectrometry

An LTQ Orbitrap XL hybrid mass spectrometry method was developed for the determination of illicit drugs and their metabolites, including amphetamine (AP), methamphetamine (MA), dimethylamphetamine (DMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), ketamine (KET), norketamine (NK), cocaine (COC) and benzoylecgonine (BE), in hair. Micropulverized extraction was employed for sample preparation using a small hair sample (2 cm piece or 0.2 mg). Recoveries of the analytes during sample preparation were estimated using fortified hair samples and ranged from 35.5% for COC to 71.7% for AP. High resolution full-scan mass spectra and unit resolution product-ion spectra were obtained with the Orbitrap analyzer and the linear ion-trap analyzer, respectively. High-resolution extracted ion chromatograms at a tolerance of 3 ppm were utilized for quantification. The analytes were identified using the product-ion spectra in combination with the accurate masses of the corresponding protonated molecules observed in the high-resolution mass spectra. Lower limits of quantification obtained from a 0.2 mg hair sample were 0.050 ng mg(-1) (MDMA, KET and BE), 0.10 ng mg(-1) (AP, MA, DMA, NK and COC) and 0.50 ng mg(-1) (MDA). Two reference materials were analyzed for verification, and segmental analysis of single strands of hair specimens from actual cases was performed.     

Determination of the residues of eleven organophosphorus insecticides in Job's-tears by gas chromatography with a nitrogen-phosphorus detector

A simplified method for determining 11 organophosphorus insecticides (dichlorvos, methamidophos, acephate, diazinon, dimethoate, chlorpyrifos, malathion, parathion, quinalphos, methidathion, and ethion) in the Chinese herbal medicine Job's-tears is described. Standards were fortified into Job's-tears (5 g) at 4 levels. The organophosphorus insecticides were extracted with dichloromethane and cleaned up with a mixture of Celite 545-activated carbon (4 + 1). The extracts were analyzed by gas chromatography using a nitrogen-phosphorus detector. Analysis of fortified Job's-tears shows average recoveries ranged from 73.90-98.70%, 86.31-93.15%, 84.92-96.22%, and 83.29-104.23% at 0.05, 0.1, 0.5, and 1.0 mg/kg levels, respectively. The minimum detectable amount ranged from 1.0 x 10(-10) to 5.0 x 10(-10) g, and the limit of quantitation for the method was 0.05 mg/kg. The method is rapid, simple, sensitive, reproducible, and applicable to the determination of these 11 organophosphorus insecticides in Job's-tears.     

Determination of benomyl, diphenyl, o-phenylphenol, thiabendazole, chlorpyrifos, methidathion, and methyl parathion in oranges by solid-phase extraction, liquid chromatography, and gas chromatography

A simple and rapid method was developed for determination of benomyl, diphenyl (DP), o-phenylphenol (OPP), thiabendazole (TBZ), chlorpyrifos, methidathion, and methyl parathion in whole oranges. These compounds were extracted from a mixture of samples and anhydrous sodium acetate with ethyl acetate. The ethyl acetate extract was concentrated and cleaned up by passing through tandem solid-phase extraction columns consisting of anion-exchange and primary/secondary amine bonded silica. The eluate was concentrated and volume was adjusted with methanol for subsequent liquid chromatography (LC) and gas chromatography (GC). Benomyl (as methyl-2-benzimidazole carbamate, MBC), DP, OPP, and TBZ residues were determined by LC with fluorescence detection. Recoveries at 3 fortified levels (0.1, 1, and 10 micrograms/g) ranged from 63.9 to 97.4%, with coefficients of variation (CVs) of 1.6 to 15.5%. Limits of detection (LODs) were 0.01 microgram/g for DP, OPP, TBZ and 0.05 microgram/g for benomyl. Chlorpyrifos, methidathion, and methyl parathion residues were determined by GC with flame photometric detection. Recoveries ranged from 90.4 to 97.0%, with CVs of 2.1 to 5.9%. LODs were 0.005 microgram/g for chlorpyrifos and methyl parathion, and 0.01 microgram/g for methidathion.     

Simultaneous determination of β‐sitosterol,campesterol, and stigmasterol in rat plasma by using LC–APCI‐MS/MS: Application in a pharmacokinetic study of a titrated extract of the unsaponifiable fraction of Zea mays L.

A liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry method was developed and validated to investigate the pharmacokinetic properties of β‐sitosterol, campesterol, and stigmasterol in rat plasma. Cholesterol‐d₆ was used as an internal standard. To avoid interference of the three phytosterols in rat plasma and minimize matrix effects, a small volume (10 μL) of 4% bovine serum albumin was used as a surrogate matrix for making calibrators and quality control samples. Rat plasma (10 μL) samples were extracted by liquid–liquid extraction with methyl tert‐butyl ether and separated on a Kinetex C₁₈ column. The detection was performed on a triple quadrupole tandem mass spectrometer in selected reaction monitoring mode using positive atmospheric pressure chemical ionization. This assay was linear over concentration ranges of 250–5000 ng/mL (β‐sitosterol), 250–5000 ng/mL (campesterol), and 50–2000 ng/mL (stigmasterol). Additionally, a second set of quality controls made in rat plasma was also evaluated against calibration curves made using the surrogate matrix. All the validation data, including the specificity, precision, accuracy, recovery, matrix effect, stability, and incurred sample reanalysis conformed to the acceptance requirements. Our method was successfully applied to study the pharmacokinetics of three phytosterols in rats.     

Residue analysis for halofuginone in sturgeon muscle by immunoaffinity cleanup and liquid chromatography

A high-performance liquid chromatography (LC) method was developed for the determination of halofuginone (HFG) in sturgeon muscle. The extracted samples were cleaned up by an immunoaffinity chromatography column that was prepared by covalently coupling polyclonal antibodies against HFG to cyanogen bromide (CNBr) activated Sepharose 4B. The eluate was evaporated to dryness, and residues were determined by LC with absorbance detection at 243 nm. Recoveries of HFG from samples fortified at 20-200 microg/kg levels ranged 74.6-81.1%, with coefficients of variation of 0.7-8.6%. The detection limit was estimated to be 10 microg/kg in a 2 g sample.     

Determination of emamectin benzoate in medicated fish feed

A method was developed to quantitate emamectin benzoate in fish feed at levels between 5 and 15 ppm. The active ingredient is extracted from 20 g medicated feed into aqueous-methanolic solvent by overnight shaking. A solid-phase extraction procedure using a 2 g C18 cartridge is then used to concentrate the active residue and remove interfering matrix components. The extracted drug and internal standard are eluted from the cartridge, evaporated to dryness, and reconstituted in methanol. A control feed sample and fortified control working standard are simultaneously prepared. Remaining interferences and sample analysis are further separated on a gradient liquid chromatographic system. Recovery of emamectin benzoate from fortified feeds ranged from 97 to 100%, with a coefficient of variation (CV) of 1.2%. Determination of emamectin benzoate in medicated feeds resulted in CVs ranging from 2.3 to 4.2% and recoveries of 88 to 98% of label claim.