HILIC-MS/MS method for the quantitation of nucleotides in infant formula and adult nutritional formula: First Action 2011.21

Official Method 2011.21 is for the quantitation of the following nucleotides: adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), uridine 5'-monophosphate (UMP), cytidine 5'-monophosphate (CMP), and inosine 5'-monophosphate (IMP) in infant formula and adult/pediatric nutritional formula. It uses hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS). Preparation of the internal standards was conducted using centrifugal ultrafiltration and the standards are AMP- (13)C10, (15)N5; GMP-(13)C10, (15)N5; UMP-(13)C9, (15)N2; and15 CMP- (13)C9, (15)N3. Data were collected by using multiple reaction monitoring of the product ions of protonated molecules of the five nucleotides generated by positive-electrospray ionization. The HILIC conditions were conducted with ammonium formate (30 mmol/L) in water (pH 2.5, adjusted with formic acid) and methanol. The LOD and LOQ of the standard solution were 0.005-0.01 and 0.01-0.03 microg/mL, respectively. Recovery data were collected for intraday and interday testing and ranged from 98.1 to 108.9% with an RSD of 0.7-5.4%. The analytical range of the method is between 0.04 to 5 microg/mL for standard solution.     

Quantitative determination of bovine caseinoglycomacropeptide in infant formulas by ultra-high-performance liquid chromatography-electrospray-ionization mass spectrometry

An ultra-high-performance liquid chromatography-electrospray ionization coupled to mass spectrometry method has been developed for determining caseinoglycomacropeptide (CGMP) in infant formulas by selected ion reaction and area monitoring modes. The present study focused on the optimization of sample pretreatment, chromatographic resolution and mass spectrometry parameters. After a simple sample pretreatment, the two genetic variants of caseinoglycomacropeptide, CGMP(A) and CGMP(B), were separated using a BEH300 C(18) column by gradient elution. The established method was extensively validated by determining the linearity (R(2)>0.999), average recovery (95.8-118.4%), inter-day precision (relative standard deviation ≤7.81%) and intra-day precision (relative standard deviation ≤6.99%) based on two scan modes. To further verify the applicability of the method, 21 brands of commercial available infant formulas were analyzed. The results showed that the present method is selective, sensitive and reliable for separating and quantifying two genetic variants (CGMP(A) and CGMP(B)) of caseinoglycomacropeptide in infant formulas with complex matrix.     

Selective determination of chromium (VI) in powdered milk infant formulas by electrothermal atomization atomic absorption spectrometry after ion exchange

A previously developed method was adapted to the selective determination of hexavalent chromium in powdered milk infant formulas. The species in reconstituted milk was separated on an ion-exchange column, Chromabond NH2, and measured by electrothermal atomization atomic absorption spectrometry. The detection limit was 1.8 micrograms/L, and the linearity range under optimized conditions was 1.8-50.0 micrograms/L. The precision values were 4.1 and 6.5% for the analytical and overall procedures, respectively. The procedure was validated by the method of standard additions (5.0, 10.0, and 25.0 micrograms/L), and the recoveries were all > 93%. The developed method is sensitive, accurate, and precise for determination of Cr(VI) in powdered milks. It was applied to the determination of Cr(VI) in 20 commercial brands, i.e., 7 infant formulas, 5 follow-up milks, and 8 dietetic milks. The values found ranged from < 10 to 75 ng/g.     

Determination of monophosphate nucleotides by capillary electrophoresis inductively coupled plasma mass spectrometry

A preliminary study of a modified microconcentric nebulizer (CEI-100, CETAC) as the sample introduction device of capillary electrophoresis inductively coupled plasma mass spectrometry (CE-ICP-MS) for the determination of monophosphate nucleotides is described. The monophosphate nucleotides studied include adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), uridine 5'-monophosphate (UMP) and inosine 5'-monophosphate (IMP). The species studied were well separated using a 70 cm length x 75 microm id fused silica capillary while the applied voltage was set at -22 kV and a 20 mmol l(-1) ammonium citrate/citric acid buffer (pH 4.0) containing 0.1% m/v cationic polymer (hexadimethrine bromide, Polybrene) was used as the electrophoretic buffer. The electroosmotic flow was reversed by flushing the fused silica capillary with 0.2% m/v Polybrene to accelerate separation. The detection limit of various species studied was in the range of 0.036-0.054 microg P ml(-1), which corresponded to the absolute detection limit of 1.1-1.6 pg P based on the injection volume of 30 nl. We determined the concentrations of nucleotides in two IG-enriched monosodium glutamates purchased from the local market. The recovery was in the range of 100-112% for various species, and the concentrations of IMP and GMP in these samples were in the range of 0.15-0.18% m/m.     

Confirmatory Analysis of Per and Polyfluoroalkyl Substances in Milk and Infant Formula Using UHPLC–MS/MS

An ultra-high performance liquid chromatography tandem mass spectrometry method was developed and validated for the sensitive determination and unambiguous confirmation of residues of per and polyfluorinated alkyl substances (PFAS) in breastmilk, retail milk and infant formulas following two sample preparation methods. Sample pre-treatment was carried out by a simplified QuEChERS method without requiring dSPE or any further clean-up. The method was validated in accordance with the requirements of Commission Decision 657/2002/EC with slight modifications. The method displayed good linearity with R² ranging from 0.9843–0.9998 for all target PFAS. The recovery and within-laboratory reproducibility of the method (n = 63) were in the range 60–121% and 5–28%, respectively. The decision limit, detection capability and limit of quantitation ranged from 30–60 ng kg⁻¹ to 40–100 ng kg⁻¹ and 5–50 ng kg⁻¹, respectively. Acceptable matrix effect values in the range −45–29% were obtained with uncertainty of measurement lower than 25% for all target PFAS. The method displays its suitability for the sensitive and high-throughput confirmatory analysis of C₄–C₁₄ PFAS in breastmilk, dairy milk and infant formulas.     

Analysis of 5'-mononucleotides in infant formula and adult/pediatric nutritional formula by liquid chromatography: First Action 2011.20

A method for the routine determination of 5'-mononucleotides (uridine 5'-monophosphate, inosine 5'-monophosphate, adenosine 5'-monophosphate, guanosine 5'-monophosphate, and cytidine 5'-monophosphate) in infant formula and adult nutritionals is described. After sample dissolution and addition of internal standard, potential interferences were removed by anion-exchange SPE followed by HPLC-UV analysis. Single-laboratory validation performance parameters include recovery (92-101%) and repeatability (1.0-2.3% RSD). The method was approved for Official First Action status by an AOAC expert review panel.     

Analysis of cytokinin nucleotides in coconut (Cocos nucifera L.) water using capillary zone electrophoresis-tandem mass spectrometry after solid-phase extraction

A method based on solid-phase extraction (SPE) and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) is described for the separation and determination of six cytokinin nucleotides in coconut water. The best CZE separation for the six cytokinin nucleotide standards was achieved using a 25 mM ammonium formate/formic acid buffer (pH 3.8) and 2% (v/v) methanol with an applied gradient separation voltage (25 kV for 32 min, and then a linear gradient to 30 kV in 5 min, finally 30 kV to the end of separation) in less than 60 min. MS/MS with multiple reaction monitoring (MRM) detection was carried out to obtain sufficient selectivity and sensitivity for the cytokinin nucleotides. The combined use of on-line sample stacking and CZE-MS/MS achieved limits of detection (LODs) in the range of 0.06-0.19 microM for the six cytokinin nucleotides at a signal-to-noise ratio of 3. Furthermore, a novel dual-step SPE procedure was developed for the pre-concentration and purification of cytokinin nucleotides using Oasis HLB and Oasis MAX cartridges. The recoveries of the cytokinin nucleotides after the dual-step SPE were in the range of 44-71%. The combination of off-line SPE, on-line sample stacking and CZE-MS/MS approach was successfully applied to screen for endogenous cytokinin nucleotides present in coconut water sample. trans-Zeatin riboside-5'-monophosphate (ZMP) was detected and quantified in coconut water by CZE-MS/MS after SPE and on-line sample stacking.     

Ion-pair high-performance liquid chromatography with diode array detection coupled to dual electrospray atmospheric pressure chemical ionization time-of-flight mass spectrometry for the determination of nucleotides in baby foods

A liquid chromatography with diode array detection coupled to dual electrospray atmospheric pressure chemical ionization time-of-flight mass spectrometry (HPLC/ESI-APCI-TOF-MS) method is described for the rapid determination of five monophosphate nucleotides (cytidine 5′-monophosphate, uridine 5′-monophosphate, adenosine 5′-monophosphate, inosine 5′-monophosphate and guanosine 5′-monophosphate) in baby foods. The method is based on the deproteinisation of foods and direct analysis of nucleotides by ion-pair HPLC using isocratic elution with a mobile phase of 5% (v/v) methanol and 95% (v/v) 0.1 M formate buffer (pH 5.5) containing 0.01 M N,N-dimethylhexylamine (DMHA) at a flow-rate of 0.7 mL min⁻¹. The HPLC was hyphenated with two different detection systems, photodiode-array (DAD) and ESI-APCI-TOF-MS in negative mode. The method was validated for linearity, detection and quantitation limits, selectivity, accuracy and precision. The recoveries obtained for spiked samples were satisfactory for all the analytes. The method was successfully applied to the analysis of nucleotides in different baby and/or functional food samples, as cereals, purees and dairy products. A study was also carried out on the stability of nucleotides in acidified dairy infant food with pasteurized yoghourt and follow-on formulae samples stored at room temperature and at 30 °C.     

Pantothenic acid (vitamin B5) in fortified foods: comparison of a novel ultra-performance liquid chromatography-tandem mass spectrometry method and a microbiological assay (AOAC Official Method 992.07)

A novel method was developed and single-laboratory validated for the determination of free pantothenic acid (vitamin B5) in a wide range of infant and adult fortified food products. The method combines simple sample preparation and chromatographic analysis using ultra-performance LC coupled to tandem MS with positive electrospray ionization. Pantothenic acid was quantified using [13C6, 15N2]-pantothenic acid as an internal standard. Calibration curves were linear between 0.08 and 1.2 microg/mL (r2 = 0.9998), and average recovery varied between 95 and 106%. The method exhibited overall RSD(r) of 1.1% and RSD intermediate reproducibility from 2.5 to 6.0% in infant formulas and cereals. Comparison of results between total and free pantothenic acid showed that the analysis of free pantothenic acid gave a good estimation of total pantothenic acid in the range of products analyzed. The method provides reliable free pantothenic acid results in a wide range of fortified foods (infant and adult nutritionals, cereal products and beverages), and shows good correlation with the microbiological method AOAC Official Method 992.07. It is a more selective, faster, and robust alternative to microbiological determination.     

Capillary electrophoresis-ion trap mass spectrometry analysis of Ziagen and its phosphorylated metabolites

A capillary electrophoresis-ion trap mass spectrometry method with a time-segment program was developed to simultaneously analyze Ziagen and its phosphorylated metabolites such as carbovir monophosphate, carbovir diphosphate, and carbovir triphosphate. By using the time-segment program, the positively charged nucleoside analog and negatively charged nucleotides were separated and detected in a single electrophoretic run. The limits of detection were less than 2 micro M for all of the analytes. Calibration curves of the compounds showed excellent linearity over the range of 2-100 micro M. The capability of the method was demonstrated by analyzing Ziagen and its phosphorylated metabolites that were spiked in cellular extracts of human peripheral blood mononuclear cells at 20 micro M levels. Some endogenous nucleotides such as adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate, were also detected in the cellular extracts.     

Deuterium in vivo labelling of cytokinins in Arabidopsis thaliana analysed by capillary liquid chromatography/frit-fast atom bombardment mass spectrometry

A method was developed for analysing the biosynthetic rate of the cytokinin class of plant hormones. Transgenic, cytokinin-overproducing Arabidopsis thaliana plants were incubated in liquid culture media enriched with 30% deuterium oxide, and incorporation into the different parts of the cytokinin molecule was analysed by capillary liquid chromatography/frit-fast atom bombardment mass spectrometry after precolumn propionylation. The sugar moieties of the cytokinins generally showed a high and independent incorporation, so the analysis in this study focused on the cytokinin base moieties. It was observed that during a 24 h incubation period almost all labelling was incorporated into the side-chain, rather than the adenine moiety. The incorporation dynamics of isopentenyladenosine-5'-monophosphate, zeatinriboside-5'-monophosphate (ZRMP) and zeatin-9-glucoside were investigated through analysis of the cytokinin base fragments in high-resolution selective ion monitoring mode. Using a fractional synthetic rate approach, the biosynthetic rate of ZRMP was determined to be 18 ng h(-1) g(-1) fresh weight, giving a turnover time of 25 h. A method for the mass isotopomer abundance analysis of the cytokinins in the zeatin family, based on selective reaction monitoring, was also developed to gain further sensitivity. Use of this technique showed that there was a higher level of enrichment in zeatin nucleotide than in the corresponding nucleoside, in agreement with the hypothesis that cytokinin nucleotides are primary products in this pathway.     

A new sample preparation and separation combination for precise,accurate, rapid,and simultaneous determination of vitamins B1, B2, B3, B5, B6, B7, and B9 in infant formula and related nutritionals by LC-MS/MS

An improved method was developed for simultaneous determination of the fortified forms of thiamine (B₁), riboflavin (B₂), nicotinamide and nicotinic acid (B₃), pantothenic acid (B₅), pyridoxine (B₆), biotin (B₇), and folic acid (B₉) in infant formulas and related nutritionals. The method employed a simple, effective, and rapid sample preparation followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). It improved upon previous methodologies by offering facile and rugged sample preparation with improved chromatographic conditions, which culminated in a highly accurate and precise method for water-soluble vitamin determination in a wide range of formulas. The method was validated over six days in ten unique matrices with two analysts and on instruments in two different labs. Intermediate precision averaged 3.4 ± 2.6% relative standard deviation and over-spike recovery averaged 100.2 ± 2.4% (n = 160). Due to refinements in sample preparation, the method had high sample throughput capacity.     

Determination of disialoganglioside GD3 and monosialoganglioside GM3 in infant formulas and whey protein concentrates by ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A method of ultra-performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UPLC-ESI-MS/MS) has been established for simultaneous determination of major disialoganglioside 3 (GD3) and monosialoganglioside 3 (GM3) in infant formulas and whey protein concentrates. Gangliosides were extracted by using the technique of Svennerholm and Fredman and then cleaned up with OASIS HLB solid-phase extraction (SPE) cartridges. The various molecular species of gangliosides were separated on an Acquity UPLC BEH C8 column and analyzed under the negative ion mode. GD3 and GM3 were rapidly quantified using internal standard (IS) method. The developed method was further validated by determining the linearity, average recovery, sensitivity (limit of quantification), and precision. The results presented high correlation coefficients (R(2) > 0.993) of the selected 16 gangliosides molecular species and provided the respective linear ranges. The limit of quantification was 0.325-0.734 mg/100 g for eight molecular species of GD3 and 0.008-0.312 mg/100 g for eight molecular species of GM3, respectively. The reasonable average recoveries (81-95%) and precision (relative standard deviation [RSD] ≤15%) were also demonstrated in three different spiked levels. This new method would be very useful in the quantitative determination of gangliosides in infant formulas and whey protein concentrates.     

Simultaneous determination of free and total choline and l‐carnitine in infant formula using hydrophilic interaction liquid chromatography with tandem mass spectrometry

A quick, simple, and reliable method was developed for the simultaneous determination of free and total choline and l‐ carnitine in infant formula employing a novel hydrophilic interaction liquid chromatography with tandem mass spectrometry method. Microwave‐assisted hydrolysis was used to shorten the hydrolysis time to only 15 min. A novel Click XIon zwitterionic stationary phase was chosen because it gave better retention, perfect resolution, and sharper symmetrical peaks compared to traditional columns. The matrix effect under different experimental conditions was evaluated by using the matrix effect factor, which employs stable isotopically labeled internal standards and is more appropriate for evaluating the matrix effect related to endogenous analytes. The accuracy and precision of the method were validated with certified reference materials. The fortified recovery values for choline and l‐ carnitine were between 85.0 and 104% with relevant standard deviations <5.0%. The established method was applied to the analysis of real infant formulas, demonstrating its applicability and feasibility.     

UV Light-induced Cross-linking of Nucleosides, Nucleotides and a Dinucleotide to the Carboxy-terminal Heptad Repeat Peptide of RNA Polymerase II as Studied by Mass Spectrometry

Abstract— We report here the results of a study to assess the usefulness of mass spectrometry as a method for rapidly locating cross-linking sites in peptides modified by UV irradiation in the presence of nucleic acid components. For this study, we selected two nucleosides (thymidine and 5-bromo-2'-deoxyuridine), two nucleotides (thymi-dine-5'-monophosphate and 5-bromo-2'-deoxyuridine-5-monophosphate) and a dinucleotide (thymidylyl-[3'→5']-2'-deoxyadenosine). The peptide picked was SPSYSPT ( l -seryl- l -prolyl- l -seryl- l -tyrosyl- l -seryl- l -prolyl- l -threonine), the heptad repeat unit found in the largest subunit of the RNA polymerase II multiprotein complex. Modified peptides were isolated by reversed-phase HPLC. Molecular mass measurements confirmed that covalent adducts had been formed. High-energy tandem collision-induced dissociation mass spectrometry pinpointed the location of cross-linking in each modified peptide as being at the tyrosine residue. These results indicate that mass spectrometry is a potentially applicable technique for location of cross-linking sites in peptides, modified by attachment of nucleosides, nucleotides and dinucleotides. Such modified peptides would be among the products expected after application of standard proteolytic and nucleolytic digestion protocols to digestion of cross-linked DNA-protein complexes.     

A highly sensitive CE-UV method with dynamic coating of silica-fused capillaries for monitoring of nucleotide pyrophosphatase/phosphodiesterase reactions

A new highly sensitive capillary electrophoresis (CE) method applying dynamic coating and on-line stacking for the monitoring of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and the screening of inhibitors was developed. NPP1 and NPP3 are membrane glycoproteins that catalyze the hydrolysis nucleotides, e.g. convert adenosine 5'-triphosphate to adenosine 5'-monophosphate (AMP) and pyrophosphate. Enzymatic reactions were performed and directly subjected to CE analysis. Since the enzymatic activity was low, standard methods were insufficient. The detection of nanomolar AMP and other nucleotides could be achieved by field-enhanced sample injection and the addition of polybrene to the running buffer. The polycationic polymer caused a dynamic coating of the silica-fused capillary, resulting in a reversed electroosmotic flow. The nucleotides migrated in the direction of the electroosmotic flow, whereas the positively charged polybrene molecules moved in the opposite direction, resulting in a narrow sample zone over a long injection time. Using this on-line sensitivity enhancement technique, a more than 70-fold enrichment was achieved for AMP (limit of detection, 46 nM) along with a short migration time (5 min) without compromising separation efficiency and peak shape. The optimized CE conditions were as follows: fused-silica capillary (30 cm effective lengthx75 mum), electrokinetic injection for 60 s, 50 mM phosphate buffer pH 6.5, 0.002% polybrene, constant current of -60 muA, UV detection at 210 nm, uridine 5'-monophosphate as the internal standard. The new method was used to study enzyme kinetics and inhibitors. It opens an easy way to determine the activities of slowly metabolizing enzymes such as NPPs, which are of considerable interest as novel drug targets.     

新型离子交换硅胶键合相的制备及评价

二甲基氯硅烷与硅胶表面反应,形成牢固的ＳｉＨ键之后,连接上活泼的中间体——烯丙基缩甘油醚作为柔软的分子臂,最后接上二乙基氨基,由此制得了新型的离子交换硅胶键合相。经漫反射红外光谱、元素分析和高效液相色谱法对键合相进行了鉴定和评价。结果表明：键合反应按预定路线进行,键合相具有较好的色谱性能。此种方法可有效地运用于无孔硅胶填料的制备。     

Determination of pesticides in milk-based infant formulas by pressurized liquid extraction followed by gas chromatography tandem mass spectrometry

An efficient and selective automated analytical method for the determination and quantification of a selected group of 12 organochlorine and organophosphorous pesticides in milk-based infant formulas has been developed. The samples were extracted by pressurized liquid extraction (PLE) and analysed using GC-MS/MS. The use of alumina as the fat retainer in the PLE extraction cell, together with the application of an injector temperature program during the GC injection process, avoided typical matrix interferences without the application of additional cleanup steps. Mean recoveries of between 70 and 110% were achieved for most of the compounds, except for chlorpyrifos methyl (50%), vinclozoline (48%), fenitrothion (56%) and procymidone (53%), with relative standard deviations ranging from 9 to 17%. Low limits of quantification were obtained for the studied compounds, from 0.01 to 2.6 μg kg⁻¹, thus guaranteeing their accurate determination within the rigorous requirements established for baby food. The validated method was applied to a pilot monitoring study in Spain. Twenty five samples of different brands of powdered infant formulas were obtained from supermarkets. Positive findings of endosulfan I, endosulfan II, fenitrothion, chlorpyrifos ethyl and bifenthrin were detected at concentrations ranging from 0.03 to 5.03 μg kg⁻¹.     

Determination of bovine lactoferrin in dairy products by ultra-high performance liquid chromatography–tandem mass spectrometry based on tryptic signature peptides employing an isotope-labeled winged peptide as internal standard

A new and sensitive determination method was developed for bovine lactoferrin in dairy products including infant formulas based on the signature peptide by ultra high-performance liquid chromatography and triple-quadrupole tandem mass spectrometry under the multiple reaction monitoring mode. The simple pretreatment procedures included the addition of a winged peptide containing the isotope-labeled signature peptide as internal standard, followed by an enzymatic digestion with trypsin. The signature peptide was chosen and identified from the tryptic hydrolyzates of bovine lactoferrin by ultra high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry based on sequence database search. Analytes were separated on an ACQUITY UPLC BEH 300 C18 column and monitored by MS/MS in seven minutes. Quantitative result bias due to matrix effect and tryptic efficiency was corrected through the use of synthetic isotope-labeled standards. The limit of detection and limit of quantification were 0.3 mg/100 g and 1.0 mg/100 g, respectively. Bovine lactoferrin within the concentration range of 10–1000 nmol L⁻¹ showed a strong linear relationship with a linear correlation coefficient (r) of >0.998. The intra- and inter-day precision of the method were RSD < 6.5% and RSD < 7.1%, respectively. Excellent repeatability (RSD < 6.4%) substantially supported the application of this method for the determination of bovine lactoferrin in dairy samples. The present method was successfully validated and applied to determination of bovine lactoferrin in dairy products including infant formulas.     

Characterization of Nucleotides and Nucleotide Sugars in Candida albicans by High Performance Liquid Chromatography–Mass Spectrometry with a Porous Graphite Carbon Column

An optimized high performance liquid chromatography electrospray ionization/tandem mass spectrometry method was developed and optimized for the determination of seven nucleotides and nucleotide sugars in the extract of Candida albicans. The chromatographic separation was performed on a porous graphitic carbon column with gradient elution using acetonitrile and aqueous ammonia acetate containing 0.1% formic acid adjusted to pH 8.8. Negative-ion mode electrospray ionization mass spectrometry was applied to improve the selectivity and sensitivity of the analysis. The calibration curves showed excellent linearity over approximately three orders of magnitude with correlation coefficients exceeding 0.9946. The relative standard deviations of measurements were smaller than 4% (n = 5) and the accuracy of the intra-day and inter-day measurements were between 95.2% and 106.7%. The extraction recovery of each component was within satisfactory ranges. The validated method was applied to characterize the nucleotides metabolic profiles of the biofilm compared to the planktonic mode to evaluate changes of energy states in different growth modes of C. albicans. The levels of nucleotides and nucleotide sugar in the biofilm model treated with 5-fluoropyrimidine, baicalein, and sodium houttuyfonate were determined to investigate their effects on nucleotide metabolism and the mechanisms of antifungal agents.