Association Between Early Amino Acid Intake and Full-Scale IQ at Age 5 Years Among Infants Born at Less Than 30 Weeks' Gestation.

Importance: An international expert committee recently revised its recommendations on amino acid intake for very preterm infants, suggesting that more than 3.50 g/kg/d should be administered only to preterm infants in clinical trials. However, the optimal amino acid intake during the first week after birth in these infants is unknown. Objective: To evaluate the association between early amino acid intake and cognitive outcomes at age 5 years. Design, Setting, and Participants: Using the EPIPAGE-2 (Epidemiologic Study on Small-for-Gestational-Age Children-Follow-up at Five and a Half Years) cohort, a nationwide prospective population-based cohort study conducted at 63 neonatal intensive care units in France, a propensity score-matched analysis was performed comparing infants born at less than 30 weeks' gestation who had high amino acid intake (3.51-4.50 g/kg/d) at 7 days after birth with infants who did not. Participants were recruited between April 1 and December 31, 2011, and followed up from September 1, 2016, to December 31, 2017. Full-scale IQ (FSIQ) was assessed at age 5 years. A confirmatory analysis used neonatal intensive care unit preference for high early amino acid intake as an instrumental variable to account for unmeasured confounding. Statistical analysis was performed from January 15 to May 15, 2021. Exposures: Amino acid intake at 7 days after birth. Main Outcomes and Measures: The primary outcome was an FSIQ score greater than -1 SD (ie, ≥93 points) at age 5 years. A complementary analysis was performed to explore the association between amino acid intake at day 7 as a continuous variable and FSIQ score at age 5 years. Data from cerebral magnetic resonance imaging at term were available for a subgroup of preterm infants who participated in the EPIRMEX (Cerebral Abnormalities Detected by MRI, Realized at the Age of Term and the Emergence of Executive Functions) ancillary study. Results: Among 1789 preterm infants (929 boys [51.9%]; mean [SD] gestational age, 27.17 [1.50] weeks) with data available to determine exposure to amino acid intake of 3.51 to 4.50 g/kg/d at 7 days after birth, 938 infants were exposed, and 851 infants were not; 717 infants from each group could be paired. The primary outcome was known in 396 of 646 exposed infants and 379 of 644 nonexposed infants who were alive at age 5 years and was observed more frequently among exposed vs nonexposed infants (243 infants [61.4%] vs 206 infants [54.4%], respectively; odds ratio [OR], 1.33 [95% CI, 1.00-1.71]; absolute risk increase in events [ie, the likelihood of having an FSIQ score >-1 SD at age 5 years] per 100 infants, 7.01 [95% CI, 0.06-13.87]; P = .048). In the matched cohort, correlation was found between amino acid intake per 1.00 g/kg/d at day 7 and FSIQ score at age 5 years (n = 775; ß = 2.43 per 1-point increase in FSIQ; 95% CI, 0.27-4.59; P = .03), white matter area (n = 134; ß = 144 per mm2; 95% CI, 3-285 per mm2; P = .045), anisotropy of the corpus callosum (n = 50; ß = 0.018; 95% CI, 0.016-0.021; P < .001), left superior longitudinal fasciculus (n = 42; ß = 0.018; 95% CI, 0.010-0.025; P < .001), and right superior longitudinal fasciculus (n = 42; ß = 0.014 [95% CI, 0.005-0.024; P = .003) based on magnetic resonance imaging at term. Confirmatory and sensitivity analyses confirmed these results. For example, the adjusted OR for the association between the exposure and the primary outcome was 1.30 (95% CI, 1.16-1.46) using the instrumental variable approach among 978 participants in the overall cohort, and the adjusted OR was 1.35 (95% CI, 1.05-1.75) using multiple imputations among 1290 participants in the matched cohort. Conclusions and Relevance: In this cohort study, high amino acid intake at 7 days after birth was associated with an increased likelihood of an FSIQ score greater than -1 SD at age 5 years. Well-designed randomized studies with long-term follow-up are needed to confirm the benefit of this nutritional approach.

Need for speed: evaluation of dilute and shoot-mass spectrometry for accelerated metabolic phenotyping in bioprocess development.

With the utilization of small-scale and highly parallelized cultivation platforms embedded in laboratory robotics, microbial phenotyping and bioprocess development have been substantially accelerated, thus generating a bottleneck in bioanalytical bioprocess sample analytics. While microscale cultivation platforms allow the monitoring of typical process parameters, only limited information about product and by-product formation is provided without comprehensive analytics. The use of liquid chromatography mass spectrometry can provide such a comprehensive and quantitative insight, but is often limited by analysis runtime and throughput. In this study, we developed and evaluated six methods for amino acid quantification based on two strong cation exchanger columns and a dilute and shoot approach in hyphenation with either a triple-quadrupole or a quadrupole time-of-flight mass spectrometer. Isotope dilution mass spectrometry with 13C15N labeled amino acids was used to correct for matrix effects. The versatility of the methods for metabolite profiling studies of microbial cultivation supernatants is confirmed by a detailed method validation study. The methods using chromatography columns showed a linear range of approx. 4 orders of magnitude, sufficient response factors, and low quantification limits (7-443 nM) for single analytes. Overall, relative standard deviation was comparable for all analytes, with < 8% and < 11% for unbuffered and buffered media, respectively. The dilute and shoot methods with an analysis time of 1 min provided similar performance but showed a factor of up to 35 times higher throughput. The performance and applicability of the dilute and shoot method are demonstrated using a library of Corynebacterium glutamicum strains producing L-histidine, obtained from random mutagenesis, which were cultivated in a microscale cultivation platform.

Dietary intake of amino acids and vitamins compared to NRC requirements in obese cats undergoing energy restriction for weight loss.

BACKGROUND: This study aimed to determine if obese cats undergoing energy restriction for weight loss would meet the National Research Council's (NRC) indispensable amino acid and vitamin recommendations when fed a purpose-formulated diet. Thirty cats were placed into one of two groups; obese (BCS 8 to 9/9; n = 16) and lean (BCS 4 to 5/9; n = 14) and included in a non-randomized retrospective observational study. Cats were fed a veterinary weight loss food during a 4-week period of weight maintenance. Obese cats (O-MAINT) refers to obese cats during this period, L-MAINT to lean cats. After this initial 4-week period, the lean cats finished the study at this time and the 16 obese cats continued and were energy restricted for a 10-week period (O-RESTRICT). Analysis for dietary concentrations of indispensable amino acid and vitamin contents were performed. Daily food intakes were used to determine minimum, maximum and average daily intakes of individual nutrients for all three groups and compared against NRC 2006 minimum requirements (MR), adequate intakes (AI) and recommended allowances (RA) for adult cats. RESULTS: Over 10 weeks, O-RESTRICT cats lost 672 g ± 303 g, representing a weight loss rate of 0.94 ± 0.28% per week. Daily intake of the majority of indispensable amino acids and vitamins was greater than the NRC 2006 recommended allowance (RA per kg ideal body weight ^0.67), except for arginine, choline, crude protein, phenylalanine plus tyrosine and threonine. All O-RESTRICT cats had minimum, average, and maximum arginine intakes less than the NRC AI. Minimum daily intake of choline was below NRC RA for all O-RESTRICT cats and below NRC MR for two. All, except one, O-RESTRICT cats had a maximum and average choline intake below RA. CONCLUSIONS: All cats remained clinically healthy and showed no clinical signs of deficiency. Dietary choline and arginine requirements of obese cats as well as health risks associated with low dietary intake during energy restriction warrant further investigation.

International Regulations on Amino Acid Use in Foods and Supplements and Recommendations to Control Their Safety Based on Purity and Quality.

We examined international regulatory developments related to the use of proteinogenic amino acids in human nutrition and concluded that the current risk-assessment practices tend to focus exclusively on setting maximum daily limits. In this brief review we argue that controlling the standards of purity and ingredient quality are the key safety issues that should be considered during risk assessment. Moreover, if maximum intake limits on amino acids are implemented, they should be defined using a well-established rationale for the health risks associated with high intakes. This would avoid setting limits that are so low that they render the dietary supplements ineffective and which, therefore, could mislead the consumer. We further suggest that there should be greater regional concordance in how the use of amino acids as ingredients is regulated and use the capacity of industry to oversee pre-competitive issues, such as standards of purity and scientific research on the safety of generic ingredients. Our arguments are based on clinical safety scientific research and oversights of amino acid purity standards conducted in the last decade by the not-for-profit international association, the International Council on Amino Acid Science.

Determination of purity values of amino acid reference materials by mass balance method: an approach to the quantification of related structure impurities.

A systematic procedure for the determination of purity values of amino acid reference materials was developed by use of mass balance method where four categories of impurities (related structure impurities (RSIs), water, organic solvent residue (OSR), and non-volatile residue (NVR)) were quantified separately. The amount of RSIs was determined using a combination of three quantification methods. To ensure metrological traceability in the determination of RSIs, at least one such impurity in each candidate amino acid reference material was quantified using liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS). Other RSIs were determined using external calibration liquid chromatography-tandem mass spectrometry (LC-MS/MS) or o-phthaldialdehyde (OPA) derivatization, followed by liquid chromatography-ultraviolet (LC-UV) measurement. As the UV absorption of most RSIs came basically from the same chromophore after OPA derivatization, a relative peak area approach was used in the LC-UV method to quantify the amount of RSIs by comparing their peak areas with that of a reference RSI. The reference RSI was pre-selected and the amount determined by LC-IDMS/MS separately. The absence of D-amino acids was confirmed using Marfey's reagent derivatization, followed by LC-UV analysis. The amounts of water, OSR, and NVR were measured using Karl Fischer coulometry, gas chromatography-mass spectrometry (GC-MS) and thermogravimetry, respectively. By using this procedure, four amino acid (L-valine, L-leucine, L-isoleucine, and L-phenylalanine) certified reference materials (CRMs) were developed from the candidate materials. The homogeneity and stability of the CRMs were demonstrated by use of LC-IDMS/MS or OPA-LC-UV method, following the principles in ISO 17034 and ISO Guide 35.Graphical abstract.

Validation of a rapid, comprehensive and clinically relevant amino acid profile by underivatised liquid chromatography tandem mass spectrometry.

Background Quantification of plasma amino acids is key to the diagnosis of inherited defects of amino acid synthesis, catabolism and transport, many of which present as clinical emergencies. The utility of this test is limited by the long analysis time and subsequent inability of laboratories to provide results in real-time. Traditionally, analysis has been performed by ion exchange chromatography (IEC) but recently there has been a move towards liquid chromatography tandem mass spectrometry (LC-MS/MS) which provides the potential for faster analysis. However, the necessity to derivatise the sample and/or utilise an ion-pair reagent, combined with lack of commercially available stable isotope internal standards (IS) has prevented laboratories fully exploiting the benefits of this methodology. We describe an underivatised LC-MS/MS method enabling patient results to be reported with an improved turnaround time (<1 h). Methods Methanolic IS was added to plasma (10 µL) to precipitate protein. Following centrifugation amino acids were analysed by LC-MS/MS using selected reaction monitoring (SRM) for each analyte and corresponding IS. Results Patient samples (n = 57) and external quality assessment (EQA) material (n = 11) were analysed and results compared with IEC. Comparable accuracy and precision were obtained with 15-min analysis time. Conclusions This method enables the analysis of a clinically comprehensive amino acid profile without the need for derivatisation/ion-pair reagents and benefitting from improved analytical quantitation through multipoint calibration and use of stable isotope IS. The analysis time is fast in comparison to IEC, improves efficiency of laboratory workflow and enables stat analysis of clinically urgent samples.

A chiral unified chromatography-mass spectrometry method to analyze free amino acids.

In this project, we aimed at analyzing native (or free) amino acids with supercritical fluid chromatography coupled to mass spectrometric detection, with modern instruments and methods, and maintaining as simple a mobile phase as possible to ensure applicability of the method. The purpose was twofold: (i) a generic method allowing for satisfactory elution of a wide range of amino acids (acidic, basic, or neutral residue) and (ii) resolution of the enantiomeric pairs. The Chiralpak ZWIX (+) and (-) stationary phases were selected as they are well-known for the enantioresolution of amino acids in liquid chromatographic modes. A wide range elution gradient, starting with a large concentration of carbon dioxide (90%) and finishing at 100% solvent (methanol containing 70 mM ammonium formate and 7% water) allowed the elution of 18 native proteinogenic amino acids out of 19 injected. In these conditions, enantioselectivity was achieved for 16 of them. The basic amino acids (arginine, histidine, and lysine) were the most difficult to elute in these conditions, resulting in rather poor peak shapes. Cysteine was never observed in any of the conditions tested. Sample application was attempted with two food supplements, tablets containing a mixture of 17 proteinogenic amino acids and capsules containing taurine and theanine that were not present in the standards used for the method development. The sample preparation method was very simple, involving dissolution of the tablets and capsules in acidified water, filtration, and dilution with methanol. Mass spectrometric detection (electrospray ionization with single-quadrupole mass detection) allowed for unambiguous identification of most amino acids, except for the leucine and isoleucine isomers that were not separated by the generic gradient. The observation of taurine and theanine also suggests that the method should be generally applicable to other native amino acids than the proteinogenic amino acids selected for the development of this method. As peak shapes and signal-to-noise ratios could still be improved, further developments are wanted to upgrade this method. Due to the wide gradient (10 to 100% co-solvent in carbon dioxide), the method cannot truly be called either supercritical fluid chromatography (SFC) or enhanced-fluidity liquid chromatography (EFLC), but should be related to "unified chromatography" (UC), joining SFC and HPLC. Graphical abstract.

Fusing metabolomics data sets with heterogeneous measurement errors.

Combining different metabolomics platforms can contribute significantly to the discovery of complementary processes expressed under different conditions. However, analysing the fused data might be hampered by the difference in their quality. In metabolomics data, one often observes that measurement errors increase with increasing measurement level and that different platforms have different measurement error variance. In this paper we compare three different approaches to correct for the measurement error heterogeneity, by transformation of the raw data, by weighted filtering before modelling and by a modelling approach using a weighted sum of residuals. For an illustration of these different approaches we analyse data from healthy obese and diabetic obese individuals, obtained from two metabolomics platforms. Concluding, the filtering and modelling approaches that both estimate a model of the measurement error did not outperform the data transformation approaches for this application. This is probably due to the limited difference in measurement error and the fact that estimation of measurement error models is unstable due to the small number of repeats available. A transformation of the data improves the classification of the two groups.

Calcium Chloride and Calcium Gluconate in Neonatal Parenteral Nutrition Solutions without Cysteine: Compatibility Studies Using Laser Light Obscuration Methodology.

There are no compatibility studies for neonatal parenteral nutrition solutions without cysteine containing calcium chloride or calcium gluconate using light obscuration as recommended by the United States Pharmacopeia (USP). The purpose of this study was to do compatibility testing for solutions containing calcium chloride and calcium gluconate without cysteine. Solutions of TrophAmine and Premasol (2.5% amino acids), containing calcium chloride or calcium gluconate were compounded without cysteine. Solutions were analyzed for particle counts using light obscuration. Maximum concentrations tested were 15 mmol/L of calcium and 12.5 mmol/L of phosphate. If the average particle count of three replicates exceeded USP guidelines, the solution was determined to be incompatible. This study found that 12.5 and 10 mmol/L of calcium and phosphate, respectively, are compatible in neonatal parenteral nutrition solutions compounded with 2.5% amino acids of either TrophAmine or Premasol. There did not appear to be significant differences in compatibility for solutions containing TrophAmine or Premasol when solutions were compounded with either CaCl2 or CaGlu-Pl. This study presents data in order to evaluate options for adding calcium and phosphate to neonatal parenteral nutrition solutions during shortages of calcium and cysteine.

Investigation of the effects of storage and freezing on mixes of heavy-labeled metabolite and amino acid standards.

RATIONALE: High-throughput metabolomics has now made it possible for small/medium-sized laboratories to analyze thousands of samples/year from the most diverse biological matrices including biofluids, cell and tissue extracts. In large-scale metabolomics studies, stable-isotope-labeled standards are increasingly used to normalize for matrix effects and control for technical reproducibility (e.g. extraction efficiency, chromatographic retention times and mass spectrometry signal stability). However, it is currently unknown how stable mixes of commercially available standards are following repeated freeze/thaw cycles or prolonged storage of aliquots. METHODS: Standard mixes for 13 C, 15 N or deuterated isotopologues of amino acids and key metabolites from the central carbon and nitrogen pathways (e.g. glycolysis, Krebs cycle, redox homeostasis, purines) were either repeatedly frozen/thawed for up to 10 cycles or diluted into aliquots prior to frozen storage for up to 42 days. Samples were characterized by ultra-high-pressure liquid chromatography/mass spectrometry to determine the stability of the aliquoted standards upon freezing/thawing or prolonged storage. RESULTS: Metabolite standards were stable over up to 10 freeze/thaw cycles, with the exception of adenosine and glutathione, showing technical variability across aliquots in a freeze/thaw-cycle-independent fashion. Storage for up to 42 days of mixes of commercially available standards did not significantly affect the stability of amino acid or metabolite standards for the first 2 weeks, while progressive degradation (statistically significant for fumarate) was observed after 3 weeks. CONCLUSIONS: Refrigerated or frozen preservation for at least 2 weeks of aliquoted heavy-labeled standard mixes for metabolomics analysis is a feasible and time-/resource-saving strategy for standard metabolomics laboratories.

Frozen, but no accident - why the 20 standard amino acids were selected.

The 20 standard amino acids encoded by the Genetic Code were adopted during the RNA World, around 4 billion years ago. This amino acid set could be regarded as a frozen accident, implying that other possible structures could equally well have been chosen to use in proteins. Amino acids were not primarily selected for their ability to support catalysis, as the RNA World already had highly effective cofactors to perform reactions, such as oxidation, reduction and transfer of small molecules. Rather, they were selected to enable the formation of soluble structures with close-packed cores, allowing the presence of ordered binding pockets. Factors to take into account when assessing why a particular amino acid might be used include its component atoms, functional groups, biosynthetic cost, use in a protein core or on the surface, solubility and stability. Applying these criteria to the 20 standard amino acids, and considering some other simple alternatives that are not used, we find that there are excellent reasons for the selection of every amino acid. Rather than being a frozen accident, the set of amino acids selected appears to be near ideal.

Quality Evaluation of Pseudostellariae Radix Based on Simultaneous Determination of Multiple Bioactive Components Combined with Grey Relational Analysis.

Pseudostellariae Radix (PR) is an important traditional Chinese herbal medicine (TCM) with vast clinical consumption because of its positive effects. However, little attention has been devoted to simultaneous analysis of its bioactive components for quality control of PR based on its different harvesting times, different growing habitats, and different processing methods. In this research, the quality of PR was evaluated based on simultaneous determination of multiple bioactive components combined with grey relational analysis (GRA). A reliable method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was established to simultaneously determine the contents of 30 components in PR, including two cyclopeptides, 12 nucleosides, and 16 amino acids. Furthermore, grey relational analysis was performed to evaluate the quality of PR samples according to the contents of these 30 components. The results showed that the quality of PR harvested in 6 August 2013, cultivated in Jurong, Jiangsu, and treated by oven drying 60 °C was better than that of other PR samples. The proposed method is useful for the overall assessment on the quality of PR, and this study provides valuable information for revealing the dynamic change laws of metabolite accumulation in PR and choosing the most suitable harvesting time and reasonable processing method of PR to obtain the best quality.

Development of C-reactive protein certified reference material NMIJ CRM 6201-b: optimization of a hydrolysis process to improve the accuracy of amino acid analysis.

To standardize C-reactive protein (CRP) assays, the National Metrology Institute of Japan (NMIJ) has developed a C-reactive protein solution certified reference material, CRM 6201-b, which is intended for use as a primary reference material to enable the SI-traceable measurement of CRP. This study describes the development process of CRM 6201-b. As a candidate material of the CRM, recombinant human CRP solution was selected because of its higher purity and homogeneity than the purified material from human serum. Gel filtration chromatography was used to examine the homogeneity and stability of the present CRM. The total protein concentration of CRP in the present CRM was determined by amino acid analysis coupled to isotope-dilution mass spectrometry (IDMS-AAA). To improve the accuracy of IDMS-AAA, we optimized the hydrolysis process by examining the effect of parameters such as the volume of protein samples taken for hydrolysis, the procedure of sample preparation prior to the hydrolysis, hydrolysis temperature, and hydrolysis time. Under optimized conditions, we conducted two independent approaches in which the following independent hydrolysis and liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) were combined: one was vapor-phase acid hydrolysis (130 °C, 24 h) and hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method, and the other was microwave-assisted liquid-phase acid hydrolysis (150 °C, 3 h) and pre-column derivatization liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The quantitative values of the two different amino acid analyses were in agreement within their uncertainties. The certified value was the weighted mean of the results of the two methods. Uncertainties from the value-assignment method, between-method variance, homogeneity, long-term stability, and short-term stability were taken into account in evaluating the uncertainty for a certified value. The certified value and the expanded uncertainty (k = 2) of CRM 6201-b are (40.0 ± 1.6) µmol kg(-1).

Surrogate matrix and surrogate analyte approaches for definitive quantitation of endogenous biomolecules.

BACKGROUND: Quantitation of biomarkers by LC-MS/MS is complicated by the presence of endogenous analytes. This challenge is most commonly overcome by calibration using an authentic standard spiked into a surrogate matrix devoid of the target analyte. A second approach involves use of a stable-isotope-labeled standard as a surrogate analyte to allow calibration in the actual biological matrix. For both methods, parallelism between calibration standards and the target analyte in biological matrix must be demonstrated in order to ensure accurate quantitation. RESULTS: In this communication, the surrogate matrix and surrogate analyte approaches are compared for the analysis of five amino acids in human plasma: alanine, valine, methionine, leucine and isoleucine. In addition, methodology based on standard addition is introduced, which enables a robust examination of parallelism in both surrogate analyte and surrogate matrix methods prior to formal validation. Results from additional assays are presented to introduce the standard-addition methodology and to highlight the strengths and weaknesses of each approach. CONCLUSION: For the analysis of amino acids in human plasma, comparable precision and accuracy were obtained by the surrogate matrix and surrogate analyte methods. Both assays were well within tolerances prescribed by regulatory guidance for validation of xenobiotic assays. When stable-isotope-labeled standards are readily available, the surrogate analyte approach allows for facile method development. By comparison, the surrogate matrix method requires greater up-front method development; however, this deficit is offset by the long-term advantage of simplified sample analysis.

[German guidelines for brain tumour imaging by PET and SPECT using labelled amino acids]. / PET- und SPECT-Untersuchungen von Hirntumoren mit radioaktiv markierten Aminosäuren.

For the primary diagnosis of brain tumours, morphological imaging by means of magnetic resonance imaging (MRI) is the current method of choice. The complementary use of functional imaging by positron emitting tomography (PET) and single photon emitting computerized tomography (SPECT) with labelled amino acids can provide significant information on some clinically relevant questions, which are beyond the capacity of MRI. These diagnostic issues affect in particular the improvement of biopsy targeting and tumour delineation for surgery and radiotherapy planning. In addition, amino acid labelled PET and SPECT tracers are helpful for the differentiation between tumour recurrence and non-specific post-therapeutic tissue changes, in predicting prognosis of low grade gliomas, and for metabolic monitoring of treatment response. The application of dynamic PET examination protocols for the assessment of amino acid kinetics has been shown to enable an improved non-invasive tumour grading. The purpose of this guideline is to provide practical assistance for indication, examination procedure and image analysis of brain PET/SPECT with labelled amino acids in order to allow for a high quality standard of the method. After a short introduction on pathobiochemistry and radiopharmacy of amino acid labelled tracers, concrete and detailed information is given on the several indications, patient preparation and examination protocols as well as on data reconstruction, visual and quantitative image analysis and interpretation. In addition, possible pitfalls are described, and the relevant original publications are listed for further information.

Amphiphilic erythromycin-lipoamino acid ion pairs: characterization and in vitro microbiological evaluation.

A series of amphiphilic ion pairs of erythromycin (ERY) with lipoamino acids (LAAs) were produced. The ion pairs were prepared by evaporation of a water/ethanol co-solution of the drug and LAA bearing an alkyl side chain of 10-16 carbon atoms. For the sake of comparison, equimolar physical mixtures were prepared by triturating ERY and the LAA in the absence of any solvent. FTIR spectroscopy confirmed the structure of ion pairs, while differential scanning calorimetry and powder X-ray diffractometry were used to assess the formation of new saline species. The solubility pattern of the coevaporates in different aqueous and organic solvents confirmed their amphiphilic properties. ERY-LAA ion pairs were submitted to an in vitro microbiological assay against different bacterial strains, both susceptible and resistant to macrolides. The presence of the LAA moiety was shown not altering the antibacterial spectrum of activity of the drug. These results can be the basis for a further evaluation of ERY-LAA ion pairs as a mean to improve the penetration of the drug inside bacterial cells and to optimize the loading of ERY in lipid-based nanocarriers.

Quantification of protein calibrants by amino acid analysis using isotope dilution mass spectrometry.

This work demonstrates that amino acid analysis based on isotope dilution mass spectrometry (IDMS) can be applied to quantify proteins having different complexities and natures. Five proteins and one decapeptide were selected for the study: C-reactive protein (CRP), beta-2-microglobulin (B2M), cystatine C (CysC), human serum albumin (HSA), Ara h1, and angiotensin I. The quantification was based on the determination of four amino acids, proline (Pro), isoleucine (Ile), valine (Val), and phenylalanine (Phe) within a working range between 5 and 100 pmol/injection of each amino acid, after 60 min digestion with HCl at 150°C. The amino acids were selected taking into account their abundance in the protein sequence and to include the more difficult to break peptide bonds. Quantification of the protein amounts calculated from each amino acid is consistent, indicating that the method is working reliably. This consistency points to a complete hydrolysis of the proteins. The trueness of the method was proven when dry mass determination after dialysis was applied to HSA and CRP and the results were compared to those from amino acid analysis. Traceability to SI was assured by extensive characterisation of the amino acid calibrants by nuclear magnetic resonance, neutron activation analysis, and Karl Fischer titration.

Investigating microwave hydrolysis for the traceable quantification of peptide standards using gas chromatography-mass spectrometry.

Over the past decade, a number of endogenous peptides and endogenous peptide analogs have been employed in therapeutics and as diagnostic markers. The use of peptides as standards for the absolute quantification of proteins has become commonly accepted. Consequently, the requirement for standard peptides traceable to the International System of Units with low associated measurement uncertainty, and for accurate methods of peptide quantification, has increased. Here we describe a method of peptide quantification involving microwave-assisted acid hydrolysis followed by gas chromatography-mass spectrometry that enables traceable quantification of a peptide by exact matching isotope dilution mass spectrometry where the total hydrolysis time required is only 3h. A solution of angiotensin I was quantified using this method, and the results were in agreement with those obtained previously using an oven hydrolysis liquid chromatography-tandem mass spectrometry method.

Certification of NIST standard reference material 2389a, amino acids in 0.1 mol/L HCl--quantification by ID LC-MS/MS.

An isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) measurement procedure was developed to accurately quantify amino acid concentrations in National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 2389a-amino acids in 0.1 mol/L hydrochloric acid. Seventeen amino acids were quantified using selected reaction monitoring on a triple quadrupole mass spectrometer. LC-MS/MS results were compared to gravimetric measurements from the preparation of SRM 2389a-a reference material developed at NIST and intended for use in intra-laboratory calibrations and quality control. Quantitative mass spectrometry results and gravimetric values were statistically combined into NIST-certified mass fraction values with associated uncertainty estimates. Coefficients of variation (CV) for the repeatability of the LC-MS/MS measurements among amino acids ranged from 0.33% to 2.7% with an average CV of 1.2%. Average relative expanded uncertainty of the certified values including Types A and B uncertainties was 3.5%. Mean accuracy of the LC-MS/MS measurements with gravimetric preparation values agreed to within |1.1|% for all amino acids. NIST SRM 2389a will be available for characterization of routine methods for amino acid analysis and serves as a standard for higher-order measurement traceability. This is the first time an ID LC-MS/MS methodology has been applied for quantifying amino acids in a NIST SRM material.