Fast and selective determination of triterpenic compounds in olive leaves by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring after microwave-assisted extraction

A method for fast and selective determination of the main triterpenic compounds present in olive leaves — oleanolic, ursolic and maslinic acids as triterpenic acids and, uvaol and erythrodiol as triterpenic dialcohols — is reported here. Quantitative isolation of the analytes has been accomplished in 5 min by microwave assistance using ethanol as extractant. Due to the medium polarity of triterpenic acids and dialcohols, different ethanol-water ratios were tested in order to select the optimum extractant composition for their solubilisation. Microwave assistance provided a significant shortening of the leaching time as compared to conventional procedures by maceration, which usually requires at least 5 h. After extraction, determination was carried out by liquid chromatography-tandem mass spectrometry (LC-MS-MS) with a triple quadrupole (qQq) mass detector without any clean-up step prior to chromatographic analysis. Highly selective identification of triterpenes was confirmed by multiple reaction monitoring (MRM) using the most representative transitions from the precursor ion to the different product ions, while the most sensitive transitions were used for MS-MS quantitation. Total analysis performed in 25 min enables the characterization of a fraction with particular interest in the pharmacological area.     

Analysis of cholesterol oxidation products by Fast gas chromatography/mass spectrometry

The aim of the present study was to set‐up a Fast gas chromatography/mass spectrometry method for the analysis of cholesterol oxidation products (COPs). A silylated mixture of seven oxysterol standards was injected into a Fast GC/MS system. A capillary GC column (10 m×0.1 mm internal diameter×0.1 μm film thickness) coated with 95% dimethyl‐ and 5% diphenyl‐polysiloxane, was used. The method gave a fast (total analysis time=3.5 min) and satisfactory resolution (R>1.2) of the COPs standards, with a good repeatability and sensitivity, similar to those of conventional GC/MS; recoveries were tested on mice liver. Fast GC/MS method suitability for COPs analysis in food was also tested on an oxidized sardine fillet, which had been previously saponified and purified by NH₂ solid‐phase extraction (SPE); a good repeatability and sensitivity was also obtained. The analytical performance of the Fast GC/MS method for the determination of COPs, together with the consequent significant reduction of the analysis time and consumables, demonstrates that Fast GC/MS represents a valid alternative to conventional GC/MS and evinces the great potential of such an analytical technique, which could be applied for both food and biological samples.     

First screening method for the simultaneous detection of seven allergens by liquid chromatography mass spectrometry

The development of a multi-method for the detection of seven allergens based on liquid chromatography and triple-quadrupole tandem mass spectrometry in multiple reaction mode is described. It is based on extraction of the allergenic proteins from a food matrix, followed by enzymatic digestion with trypsin. The chosen marker peptides were implemented into one method that is capable of the simultaneous detection of milk, egg, soy, hazelnut, peanut, walnut and almond. This method has been used to detect all seven allergenic commodities from incurred reference bread material, which was baked according to a standard recipe from the baking industry. Detected concentrations ranged from 10 to 1000 μg/g, demonstrating that the mass spectrometric based method is a useful tool for allergen screening.     

Metabolomic analysis of saponins in crude extracts of Quillaja saponaria by liquid chromatography/mass spectrometry for product authentication

Analysis of 50% aqueous methanolic extracts of bark of Quillaja saponaria Molina (quillaja) by liquid chromatography/mass spectrometry (LC/MS), using negative ion electrospray, revealed over 100 saponins. The majority could be assigned to known structures or generalised variations of these from the product ion spectra obtained by serial mass spectrometry in a quadrupole ion trap mass spectrometer. Ten saponins contained a fatty acid domain terminated with both a pentose and deoxyhexose unit, a feature thus far only reported in QS-III. Twenty saponins were based on a hydroxylated derivative of quillaic acid, whereas only six 22beta-hydroxyquillaic acid saponins have been described. The occurrence of pairs of saponins differing only by the presence of a rhamnose or xylose unit in the C-3-substituted saccharide was readily observed in two-dimensional mass maps, and these showed the presence of the unreported 'rhamnose partner' of QS-III. However, one sample labelled as Q. saponaria appeared to lack all saponins containing rhamnose in the C-3 saccharide. Methods to authenticate saponin extracts of quillaja by LC/MS are suggested based on the general metabolomic profile, the occurrence of specific major saponins covering known structural variations, or the presence of saponins containing the unusual fatty acid domain, revealed by neutral loss analysis.     

Rapid screening for bisbibenzyls in bryophyte crude extracts using liquid chromatography/tandem mass spectrometry

A simple and rapid qualitative liquid chromatography-diode-array detection/tandem mass spectrometry (LC-DAD/MS/MS) method was developed and validated for screening bisbibenzyl compounds in bryophyte crude extracts at sub-ppm levels. After simple extraction with ethanol and analyte concentration with diethyl ether, the extracts were subjected to LC-DAD/MS/MS analysis. The overall instrument turnaround time was 50 min to obtain baseline separation of bisbibenzyl isomers in bryophytes. MS full scan, MS/MS precursor ion scan and MS/MS product ion scan modes were used for the screening. The bisbibenzyl standards studied gave limits of detection (LODs) at or below 10 ng/mL. The results also indicated that the method had acceptable precision to be used on a day-to-day basis for qualitative identification. The bisbibenzyl types, i.e. one biphenyl ether bond (A-type), two biphenyl ether bonds (B-type), one biphenyl ether and one biphenyl bond (C-type), or other biphenyl types can be differentiated by their ESI-MS/MS product profiles, and the number of alkoxyl substituents can also be identified. The linkage sites of biphenyl and biphenyl ether bonds cannot be identified for an unknown bisbibenzyl solely from its mass spectra. This system was used to support three screening assays of bryophytes including Marchantia polymorpha L., Ptagiochasm intermedium L. and Asterella angusta, which were collected from different places in China. From them, 7/12, 8/5 and 8/9 confirmed/unconfirmed bisbibenzyls were identified, respectively, based on their MS/MS data, UV spectra and the retention behavior. The screening method considerably reduced the time and the cost for the qualitative analyses, and the structure-fragmentation-UV relationships will facilitate the high-throughput screening (HTS) of bisbibenzyl compounds in bryophytes. It is also intended as a simple and convenient way for the determination of other structural families of natural products.     

液相色谱-串联质谱法测定海参和海胆中的单唾液酸神经节苷脂

建立了液相色谱-四极杆串联质谱法定量检测海参和海胆中单唾液酸神经节苷脂的分析方法。采用Svennerholm法从海胆或海参样品中提取神经节苷脂,经C8固相萃取柱净化,采用APS-2 NH₂柱(150 mm×2.1 mm, 3 μm),以乙腈和50 mmol/L乙酸铵溶液(pH 5.6)为流动相,梯度洗脱。样品中每种成分的定量在多反应监测模式下进行。该方法具有极高的灵敏度,定量限可低至纳克级。非硫酸酯化单唾液酸神经节苷脂(NMG)和硫酸酯化单唾液酸神经节苷脂(SMG)在1～40 ng进样量范围内呈现良好的线性关系;定量结果显示所测海参样品中美国红参的NMG含量最高,海胆样品中紫海胆的SMG含量最高;海胆中总的单唾液酸神经节苷脂含量(4.30～6.40 mg/g)明显高于各海参样品(8～131 μg/g)。该方法稳定可靠,适合海胆和海参中微量单唾液酸神经节苷脂的定量分析。     

Qualitative and quantitative analysis of lipid extracts from rabbit meat by gas chromatography/mass spectrometry.

Analyses of lipid extracts from rabbit meat were carried out by gas chromatography/mass spectrometry (GC/mS) using both electron and chemical ionisation. Ten rabbit carcasses were randomly acquired on the market, from different farms; for each of them muscular tissues from hindleg and breast were analysed. The lipid fractions were extracted, separated and hydrolysed. The fatty acid fractions were derivatised by 2,2-dimethoxypropane. The GC/mS data obtained using electron ionisation (EI) did not allow the complete characterisation of the fatty acid fraction, and for this reason chemical ionisation (CI) was employed using acetonitrile as reactant gas. The data thus obtained show that, for both samples of rabbit tissue, the mean abundance ratio of plasma cholesterol lowering fatty acids and plasma cholesterol elevating fatty acids (PCL/PCE), taken as a parameter describing a desirable lipid uptake, is 2.2 +/- 0.3, significantly higher than the values reported for other meats (0.8-1. 8). These data, together with the high concentration of (n-6) fatty acids, provide a good indication of the high nutritional value of rabbit meat.     

Stable isotope dilution analysis of N-acetylaspartic acid in urine by liquid chromatography electrospray ionization tandem mass spectrometry

N-acetylaspartic acid (NAA) is a specific urinary marker for Canavan disease, an autosomal recessive leukodystrophy. We developed a 'dilute and shoot' stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of NAA in urine. Deuterated internal standard d(3)-NAA was added to untreated urine and the mixture was injected into the LC-MS/MS system operated in the negative ion mode. Chromatography was carried out on a C(8) minibore column using 50% acetonitrile solution containing 0.05% formic acid at a flow rate of 0.25 mL/min. The retention time was 1.6 min and the turnaround time was 2.2 min. NAA and d(3)-NAA were analyzed in multiple reaction monitoring mode. Calibrators and quality control samples were prepared in pooled control urine. The assay was linear up to 2000 micromol/L with limit of quantification at 1 micromol/L (S/N = 12). Interassay and intraassay coefficients of variation were less than 7% and recovery at three different concentrations was 98.9-102.5%. The LC-MS/MS method for NAA as described involves no extraction and no derivatization, showed no interference and gave excellent recovery with low variability and short analytical time. The method was successfully applied for the retrospective analysis of urine from 21 Canavan disease cases.     

Development of gas chromatographic method for determination of cholesterol oxides in meat products

Summary A method of extraction, purification and gas chromatographic analysis of five cholesterol oxidation products (COPs) (7β-hydroxycholesterol, 5α, 6α-epoxycholesterol, 25-hydroxycholesterol, 7-ketocholesterol and cholestane-3β, 5α, 6β-triol) has been developed. The method is aimed at the determination of COPs in meat and meat products, in particular dry sausages, where large differences in the content of cholesterol and its oxidation derivatives may occur. Linearity of response of trimethylsilyl ethers was defined. The minimum concentration tested was 2.5 μg g⁻¹ in the injected solution. The main feature of the present work is the addition of a purification step of unsaponifiables by SPE on silica cartridges. Extraction recoveries of COP standard mixtures, performed with or without the SPE step, are evaluated. The introduction of the clean-up step results in high purity samples without further losses in the entire process.     

液相色谱–大气压化学电离串联质谱法测定油脂中9种胆固醇氧化物

建立一种准确测定油脂中9种胆固醇氧化物含量的同位素稀释液相色谱-串联质谱测定方法。向油脂样品中加入无水乙醇和60%氢氧化钾溶液,在室温下避光皂化22 h,使用乙醚-石油醚混合液(1∶1)作为提取溶液,采用液-液萃取方式提取目标物,再使用硅胶柱进行净化,采用5 mmol/L乙酸铵溶液与甲醇-乙腈混合液(1∶1)为双流动相梯度淋洗,使用Agilent ZORBAX SB-C18色谱柱分离,在大气压化学电离(APCI)正离子模式下,采用多反应监测模式内标法定量测定。在10~500μg/L范围内,9种胆固醇氧化物的质量浓度与色谱峰面积线性良好,相关系数(r2)均大于0.995,检出限均为0.5 mg/kg,在1,5 mg/kg两个添加水平时,加标回收率为71.2%~94.4%,相对标准偏差小于10%(n=6)。该方法满足动植物油脂中胆固醇氧化物含量测定需要。     

高效液相色谱-串联质谱法检测花生中的黄曲霉毒素B_1

应用高效液相色谱-电喷雾串联四极杆质谱联用系统(HPLC-MS/MS),在多反应离子检测方式(MRM)下,对花生中的黄曲霉毒素B1进行检测.对花生中黄曲霉毒素B1的提取、净化、液相分离及串联质谱等相关检测参数进行了优化研究.用V(甲醇):V(水)=6:4提取,OASIS HLB SPE小柱净化,定容过滤.采用V(甲醇):V(水)(含体积分数0.1%甲酸)=7:3为流动相,前级离子313.0,二级离子241.1、269.1,ESI正离子方式检测,在3.3 min出峰.结果表明,在ESI正离子模式下,黄曲霉毒素B1在其线性定量范围0.1～50μg/kg内,相关系数达到0.9999,检出限为0.03μg/kg,最低定量限为0.1μg/kg.低、中、高浓度添加回收率范围为93%～105%.     

Rapid quantification of iridoid glycosides analogues in the formulated Chinese medicine Longdan Xiegan Decoction using high-performance liquid chromatography coupled with mass spectrometry [corrected

Longdan Xiegan Decoction (LXD) is a formulated preparation composed of 10 ingredient herbs, with iridoids as the main bioactive components. In this study, a rapid, simple and reliable method of simultaneous determination of four iridoid glycosides in LXD using high-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (MS) was first developed and validated. The four iridoid glycosides references were isolated from LXD extract and purified using a preparative HPLC chromatography. The sample preparation for quantification comprised of a simple ultrasonic extraction and the satisfactory chromatographic separation of the four structurally similar iridoid glycosides was effected in less than three minutes on a CAPCELL PAK C(18) MGII column (3 microm, 100 mm x 2.0 mm), using an elution system of 10% methanol and their concentrations in different batches of LXD and ingredient herbs were simultaneously determined by HPLC-MS/MS using a multiple reaction monitoring (MRM) mode. The method was validated with respect to the overall intra- and inter-day variation (RSD less than 8%) and the limits of quantification for the four iridoid glycosides were 35, 20, 37 and 33 ng/mL, respectively.     

Application of selected ion monitoring to the analysis of triacylglycerols in olive oil by high temperature-gas chromatography/mass spectrometry

The analysis of the triacylglycerol (TAG) composition of oils is a very challenging task, since the TAGs have very similar physico-chemical properties. In this work, a high temperature-gas chromatographic method coupled to electron ionization-mass spectrometry (HT-GC/EI-MS), in the Selected Ion Monitoring (SIM) mode, method was developed for the analysis of TAGs in the olive oil; this is a method suitable for routine analysis. This method was developed using commercially available standard TAGs. The TAGs studied were separated according to their equivalent carbon number and degree of unsaturation. The peak assignment was carried out by locating the characteristic fragment ions having the same retention time on the SIM profile such as [RCO+74]⁺ and [RCO+128]⁺ ions, due to the fatty acyl residues on sn-1, sn-2 and sn-3 positions of the TAG molecule and the [M−OCOR]⁺ ions corresponding to the acyl ions. The developed method was very useful to eliminate the interferences that appeared in the mass spectrum since electron ionization can prevent satisfactory interpretation of spectra.     

气相色谱-三重四极杆质谱法同时测定烟丝中73种香气物质

建立了气相色谱-三重四极杆质谱(GC-MS/MS)同时测定烟丝中73种香气物质的分析方法。样品用无水乙醚振荡提取,提取液经过滤后直接进入DB-5MS色谱柱(30 m×0.25 mm,0.25μm)分离,通过优化质谱参数,可有效降低复杂基质和重叠峰的干扰,同时采用多反应监测(MRM)模式测定,内标法定量。结果表明,目标物在各自的线性范围内线性关系良好(r~20.99),低、中、高3个水平的加标回收率为70.0%~122.3%,相对标准偏差(RSD)为1.6%~20.4%。该方法具有前处理简单、准确灵敏的特点,适用于烟丝样品中73种香气物质的检测。     

Extraction and analysis of pharmaceuticals in polluted sediment using liquid chromatography mass spectrometry

A multi-residue method for the extraction and clean-up of sediment samples was developed for the analysis of pharmaceutical residues. Sediment samples were collected in the proximity of sewage water plant in Stockholm, Sweden. Target analytes were the basic β-blocker propranolol, the neutral neuroleptic carbamazepine and the acidic anticoagulant warfarin, the painkiller diclofenac and the lipid regulator gemfibrozil. The extraction solvent was optimised with regard to pH and organic modifer. Extraction and clean up were performed with liquid-liquid extraction and ultra-sonication followed by solid-phase extraction. One extraction solvent, containing acetone/McIlvaine buffer pH4, provided satisfactory extraction for all substances. LC/MSMS in the MRM mode was used for determination. The recoveries of the extraction and clean-up steps were 60–75% (±2–8%) and LOQs were in the range 0.4–8?ng/g sediment (dry weight). The pharmaceuticals found in the sediment samples were propranolol and carbamazepine, representing substances with basic and neutral properties. Additionally, the samples were analysed with LC/QTOF for verification with the use of accurate mass measurement in the full-scan mode. Pharmaceuticals not represented in the original method were looked for. Non-target pharmaceuticals found using the LC/QTOF system were the basic β-blocker metoprolol and the acidic painkiller naproxen.     

Sensitive determination of phloroglucinol in human plasma by liquid chromatography-tandem mass spectrometry

Summary A sensitive and selective liquid chromatographic method coupled with tandem mass spectrometry (LC-MS-MS) was developed for the quantification of phloroglucinol in human plasma. Resorcinol was used as internal standard, with plasma samples extracted using ethyl acetate. A centrifuged upper layer was then evaporated and reconstituted with mobile phase. The reconsituted samples were injected into a C₁₈ XTerra MS column (2.1×100 mm) with 3.5 μm particle size. The analytical column lasted for at least 500 injections. The mobile phase was 15% acetonitrile (pH 3.0), with a flow rate at 100 μL min¹. The mass spectrometer was operated in positive ion mode using electrospray ionization. Using MS-MS in multiple reaction monitoring (MRM) mode, phloroglucinol was detected without severe interferences from the plasma matrix. Phloroglucinol produced a parent molecule ([M+H]⁺) atm/z 127 and a corresponding product ion atm/z 8l. Detection of phloroglucinol in human plasma was accurate and precise, with quantification on limit at 0.5 ng mL¹. The method has been successfully applied to a study of phloroglucinol in human specimens.     

Determination of heterocyclic aromatic amines (HAA) in commercially available meat products and fish by high performance liquid chromatography—Electrospray tandem mass spectrometry (HPLC-ESI-MS-MS)

Summary Ten heterocyclic aromatic amines (HAA) (1)–(10) were analyzed in commercially available meat products and fish. After sample preparation by Extrelut treatment and subsequent solid phase extraction applying propylsulphonic and C₁₈ silica cartridges, HPLC-ESI-MS-MS using selected reaction monitoring (SRM) and d₃-PhIP and d₃-MeIQx as internal and external standards, respectively, revealed the widely distributed presence of PhIP (8) and MeIQx (4), ranging from 0.1 to 5.3 ng g⁻¹ and 0.1 to 5.2 ng g⁻¹, respectively. Lower amounts were found for 4,8-DiMeIQx (5) and 7,8-DiMeIQx (6), ranging from 0.2 to 2.0 ng g⁻¹ and 0.1 to 0.2 ng g⁻¹, respectively. The other HAA under study, i.e. IQ, MeIQ, 4,7,8-TriMeIQx, Glu-P-1, and Glu-P-2 were not determinable under the experimental conditions used (determination limit 0.1 ng g⁻¹).     

Nitrite oxidation in ion chromatography-electrospray ionization-tandem mass spectrometry (IC-ESI-MS/MS)

Nitrite anions are formed in the human body and in the natural environment as intermediate chemical compounds during the reduction of nitrate, a ubiquitous anthropogenic contaminant introduced into the environment primarily through fertilizer use. Multiple reaction monitoring (MRM) in ion chromatography-electrospray ionization-tandem mass spectrometry (IC-ESI-MS/MS) is a promising new technique for quantifying and confirming the identity of anions in complex aqueous mixtures. In this article, we present the results of a short investigation devised to: (1) compare the signal generated by the MRM transitions for nitrite with those for nitrate, (2) isolate the source of the signal from these MRM transitions occurring within the IC-ESI-MS/MS instrument and (3) assess the relationship between the observed MRM signals for nitrite. The MRM transitions used in this study were m/z 62 (NO(3)(-))→m/z 46 (NO(2)(-)) and m/z 46 (NO(2)(-))→m/z 46 (NO(2)(-)). Results of the investigation revealed the association of both MRM transitions with the nitrite chromatographic peak, indicating the occurrence of nitrite oxidation to nitrate at the ESI interface before the first quadrupole. Calibrations for both MRM signals, as well as their sum, were found to be linear. However, the ratio of m/z 62→m/z 46 to m/z 46→m/z 46 (indicating an extent of oxidation) ranged from 35 to 56% over a nitrite concentration range of 10 to 100 ppm, showing no clear trend associated with concentration.     

High performance liquid chromatography —Electrospray tandem mass spectrometry (HPLC-ESI-MS-MS) for the analysis of heterocyclic aromatic amines (HAA)

Summary Sensitive and selective detection of the sixteen most abundant heterocyclic aromatic amines (HAA) has been achieved by application of high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS-MS) in combination with selected reaction monitoring (SRM). Detection limits between 0.1 and 50 ng mL^–1 were established by use of HAA model solutions.     

Reversed‐phase separation parameters for intact proteins using liquid chromatography with triple quadrupole mass spectrometry

The separation of intact proteins is inherently more complex than that of small molecules using reversed‐phase liquid chromatography. The goal of this work was to determine a reasonable set of operational parameters (a recommended starting point for other analysts) for the separation of intact proteins and their detection by triple quadrupole mass spectrometry. Although protein separations have been studied for many years, the direct detection of intact proteins with mass spectrometry requires special considerations of mobile phase additives to achieve efficient separation and sensitive detection. Myoglobin, cytochrome c, lactalbumin, lysozyme, and ubiquitin were used as model analytes to investigate chromatographic method development using a triple quadrupole mass spectrometer and detection by multiple reaction monitoring. Chromatographic parameters including the concentration of trifluoroacetic acid, flow rate, gradient slope, temperature, mobile phase composition, and stationary phase chemistry were evaluated. Protein charge state profiles were also monitored for temperature and modifier effects. An optimized method using 0.2 mL/min flow rate, 15% gradient slope, and 75°C with a combined trifluoroacetic acid and formic acid modified mobile phase was developed.