Determination Of Carnitine Research Articles

Evaluation of Serum Amino Acid and Carnitine Profile in Dogs with Transmissible Venereal Tumor

The presented study aimed to reveal the changes in serum amino acid and carnitine profiles in dogs with transmissible venereal tumor (TVT). The study material comprised 40 female dogs ranging in age from 3 to 5 years. The dogs were divided into two groups based on genital organ examinations. Group 1 (n=20) consisted of healthy dogs, while Group 2 (n=20) consisted of TVT-positive dogs. The definite diagnosis of transmissible venereal tumor was made based on the detection of intracytoplasmic vacuoles and numerous mitotic figures in the collected vaginal smear samples. Blood samples were taken from dogs in both study groups and serum was obtained. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for the determination of carnitine and amino acid profiles. The obtained data were compared using independent samples t-test. The serum amino acid profiles of Lysine, Aspartic Acid, Tyrosine, Asparagine, Alanine, Arginine, Citrulline, Glutamic Acid, Glycine, Methylglutaryl, Phenylalanine, and Ornithine were found to be lower in the TVT group (P

Peripheral blood level of carnitine in neonates and its influencing factors

Objective To determine the peripheral blood level of carnitine in neonates and to analyze its influencing factors. Methods All newborns (n=46 250) born in Hunan provincial maternal and child healthcare hospital from 2015 to 2017 were included in this study, except for those primary carnitine deficiency and motherhood carnitine deficiency. Heel prick blood samples were collected 48 hours after birth for determination of carnitine by tandern mass spectrometry. All subjects were grouped according to gender, gestational age, birth weight, number of pregnancies, sample collecting time and whether to be hospitalized. The level of carnitine was analyzed with Mann-Whitney U test, Kruskal-Wallis H test and mutiple linear regression. Results The free carnitine levels of 46 250 neonates were ranged from 5.21 to 56.32 μmol/L, with a geometric mean of 16.05(15.99 to 16.10)μmol/L; males (n=24 375) and females (n=21 875) were 16.68 μmol/L and 15.38 μmol/L; multiple pregnancies (n=3 552) and single pregnancies (n=42 698) were 19.22 μmol/L and 15.81 μmol/L, respectively; hospitalized patients (n=6 409 cases) and non-hospitalized patients (n=3 9841 cases) were 20.10 μmol/L and 15.48 μmol/L respectively. The levels of serum free carnitine were higher in male, multifetal and hospitalized patients (Z=23.739, 30.260, 51.336, P<0.05). Comparing the concentration of free carnitine in 8 groups with different gestational age, 7 groups with different birth weight and 5 groups with different blood collecting age, the differences were statistically significant (H=2369.832, 3032.716, 967.440, P<0.05). Multivariate linear regression showed that all the six factors entered the regression equation, and the sequence was hosp-italization, birth weight, gestational age, number of pregnancies, sex, blood collection age. Conclusions The peripheral blood level of carnitine in neonates is affected by many factors, such as whether to be hospitalized, birth weight, gestational age and so on. Key words: Carnitine/BL; Infant, newborn; Factor analysis, statistical

Determination of carnitine and acylcarnitines in human urine by means of microextraction in packed sorbent and hydrophilic interaction chromatography–ultra-high-performance liquid chromatography–tandem mass spectrometry

A method using semi-automatic microextraction by packed sorbent (eVol®-MEPS) and hydrophilic interaction chromatography–ultra-high-performance liquid chromatography–tandem mass spectrometry (HILIC–UHPLC–MS/MS) was described for the simultaneous determination of carnitine and acylcarnitines in human urine. The optimal conditions of MEPS extraction were obtained using C2 of M1 (C8+SCX) phase as a sorbent. Chromatographic separation of the analytes was achieved within 2.5min on Acquity UPLC BEH HILIC column using a gradient elution program with water containing 5mM ammonium acetate and acetonitrile as the mobile phase. The detection was performed on a triple-quadrupole tandem mass spectrometer in a positive ion mode via electrospray ionization (ESI).The linearity of the calibration curves for all compounds was found over a range from 0.1ng/mL to 500ng/mL. The method afforded satisfactory results in terms of sensitivity, specificity, precision, accuracy, recovery as well as stability of the analyte under various conditions. The method was used successfully for determination of carnitine and acylcarnitines in human urine.

Quantification of plasma carnitine and acylcarnitines by high-performance liquid chromatography-tandem mass spectrometry using online solid-phase extraction

Carnitine is an amino acid derivative that plays a key role in energy metabolism. Endogenous carnitine is found in its free form or esterified with acyl groups of several chain lengths. Quantification of carnitine and acylcarnitines is of particular interest for screening for research and metabolic disorders. We developed a method with online solid-phase extraction coupled to high-performance liquid chromatography and tandem mass spectrometry to quantify carnitine and three acylcarnitines with different polarity (acetylcarnitine, octanoylcarnitine, and palmitoylcarnitine). Plasma samples were deproteinized with methanol, loaded on a cation exchange trapping column and separated on a reversed-phase C8 column using heptafluorobutyric acid as an ion-pairing reagent. Considering the endogenous nature of the analytes, we quantified with the standard addition method and with external deuterated standards. Solid-phase extraction and separation were achieved within 8 min. Recoveries of carnitine and acylcarnitines were between 98 and 105 %. Both quantification methods were equally accurate (all values within 84 to 116 % of target concentrations) and precise (day-to-day variation of less than 18 %) for all carnitine species and concentrations analyzed. The method was used successfully for determination of carnitine and acylcarnitines in different human samples. In conclusion, we present a method for simultaneous quantification of carnitine and acylcarnitines with a rapid sample work-up. This approach requires small sample volumes and a short analysis time, and it can be applied for the determination of other acylcarnitines than the acylcarnitines tested. The method is useful for applications in research and clinical routine.

Rapid quantitative analysis of carnitine and acylcarnitines by ultra-high performance–hydrophilic interaction liquid chromatography–tandem mass spectrometry

l-Carnitine and its acyl esters (acylcarnitines) play an important role in the metabolism of fatty acids. However, most of the present methods for the quantitative analysis of acylcarnitines have restrictions both in sample preparation and in chromatographic separation. Herein we present a validated method for determination of carnitine and eleven acylcarnitines in human serum and rat tissue biopsies by using ultra-high performance–hydrophilic interaction liquid chromatography–tandem mass spectrometry (UHP–HILIC–MS/MS). The procedure uses minimal sample preparation including only addition of organic solvent, labeled internal standard, incubation and centrifugation. The separation is performed without derivatization or addition of ion-pairing reagent within 7min on a hydrophilic interaction liquid chromatographic column with mass spectrometric detection. The method is linear in response over the concentration range from 20 to 600ng/ml for carnitine and acetylcarnitine and 5–200ng/ml for the other acylcarnitines, with correlation coefficients higher than 0.994. Recoveries were higher than 88% for most of the compounds. Limits of detection were 5ng/ml for carnitine and acetylcarnitine and approximately 0.5ng/ml for other acylcarnitines. The method was applied to the analysis of serum and tissue samples.

Capillary electrophoresis with contactless conductivity detection for the determination of carnitine and acylcarnitines in clinical samples

A capillary electrophoresis method with contactless conductivity detection was developed for the quantification of carnitine and six acylcarnitines in plasma and urine samples. The running buffer employed consisted of 500 mmol/L acetic acid, 1.0 mmol/L hydroxypropyl-β-cyclodextrin and 0.05% Tween at a pH of 2.6. Under these conditions, the isomeric valproyl- and octanoyl-carnitines could be distinguished. The linearity was in the range from 5.0 to 200.0 μmol/L with correlation coefficients between 0.9992 and 0.9997. The limits of detection were between 1.0 and 3.2 μmol/L. Intra- and inter-day precisions as %RSD were better than 10%. The method allows for direct determination without derivatisation or extraction processes. The method was applied for the quantification of carnitine and acetylcarnitine in plasma pre- and post-exercise, and to measure valproylcarnitine in plasma and urine of patients undergoing valproate therapy.

Determination of carnitine in food and food supplements by capillary electrophoresis with contactless conductivity detection

A CE method with capacitively coupled contactless conductivity detection was developed and tested for the quantification of carnitine in different types of foodstuffs, namely fruit juices, milk, yogurt, cheese, red meat and chicken meat. Sample preparation was minimal as chemical or enzymatic conversion of the analyte is not necessary with the non-UV-absorbing compound when conductivity detection is employed. A 500 mmol/L acetic acid solution at pH 2.6 with 0.05% Tween-20, was used as the optimized running buffer. The analysis time was approximately 4 min. Linearity was achieved for the concentration range of 5-500 micromol/L with a correlation coefficient of 0.9996. The LOD (3 signal/noise) was determined as 2.6 micromol/L. Intra- and inter-day variabilities were less than 10% for both migration time and peak area, indicating a good precision of the method.

Determination of carnitine, its short chain acyl esters and metabolic precursors trimethyllysine and γ-butyrobetaine by quasi-solid phase extraction and MS/MS detection

For the investigation of the metabolism and biosynthesis of carnitine, sensitive determination of carnitine and its metabolic precursors, trimethyllysine and γ-butyrobetaine, is required. We present here a new simplified method for the analysis of carnitine, its acetyl- and propyl esters, as well as trimethyllysine and γ-butyrobetaine without need for derivatization reactions by means of normal-phase LC and electrospray ionization tandem mass spectrometry. The limits of quantification were between 5nM for acetyl carnitine and 70nM for carnitine. Relative standard deviations in a fivefold determination of standard solutions were between <2% for carnitine and <10% for trimethyllysine. Quantifying the formation of deuterated carnitine from deuterated γ-butyrobetaine, this method is also suitable for the determination of the activity of γ-butyrobetaine dioxygenase in tissues.

Determination of free and total carnitine in plasma by an enzymatic reaction and spectrophotometric quantitation spectrophotometric determination of carnitine

Objectives Carnitine initiates the beta-oxidation of fatty acids and its deficiency is a problem in several metabolic diseases. This work describes a validated quick and simple enzymatic assay for the determination of free and total carnitine in plasma. Methods Carnitine reacts with acetyl-CoA catalized by carnitine acetyltransferase. The coenzyme A liberated combines with 5,5′-dithiobis-(2-nitrobenzoic acid) and forms thiophenolate ion, measured spectrophotometically at 412 nm. The method requires precipitation of proteins and the alkaline hydrolysis of acylcarnitines for total carnitine. Results The detection limit is 1.7 μmol/L in plasma and inter- and intra-day imprecision is less than 5%. The recovery of spiked plasma samples is 97%. The method was tested with an inter-laboratory assay, yielding excellent correlation ( r 2 = 0.994), and it was applied to the determination of normal values of carnitine in plasma. Conclusions A reliable spectrophotometric method has been validated with very good precision, with simple instrumental and easy sample preparation.

Current Methods for Determination of L-Carnitine and Acylcarnitines

L-Carnitine as endogenous compound plays an important role within several metabolic pathways and a deficiency of L-carnitine can cause adverse effects in physiological and/or mental state of health and disease. The prevention of diseases related to carnitine deficiency requires, first of all, the exact determination of L-carnitine and its esters in biological material at pmol/cm3 level. A series of analytical procedures based on biochemical assays as well as on physical methods are available today. Determination of free and total carnitine is sometimes sufficient for a clinical diagnosis, but in most cases, such as in newborn screening for genetic disorders, detailed qualitative and quantitative L-carnitine/acylcarnitine profiling is needed. Technological progress has also revolutionized the determination of carnitines. Today, comprehensive and diagnostically relevant information can be obtained by mass spectrometry. An overview is given of the technical and methodological developments in carnitine analysis and some applications, such as in neonatal screening, diabetes mellitus, and cardiomyopathy.

Capillary electrophoresis determination of carnitine in food supplements

l-Carnitine is a substance natural for human body which transfers fatty acids to the place of burning—mitochondria and aids the transformation of fats into energy and this way supports overweight reduction and immediate physical performance, increases resistance from physical load and protect heart from overload. In this study are described newly developed electrophoretic methods (ITP, CZE with direct and/or indirect UV detection) for carnitine determination in various samples. The results were compared with results obtained by validated HPLC method. All of these methods gave comparable results. The detection limits of the electrophoretic methods were between 2.4 and 4.7 μg/ml, reproducibility (relative standard deviation, RSD%) was between 1.2 and 4.4% and recoveries were between 91 and 113% in different samples. The shorter analysis and low running cost are the main advantages of CE methods.

Determination of l-carnitine in food supplement formulations using ion-pair chromatography with indirect conductimetric detection

A novel method for the determination of l-carnitine in food supplement formulations was developed and validated, using ion-pair chromatography with indirect conductimetric detection. The chromatographic method was based on a non-polar (C 18) column and an aqueous octanesulfonate (0.64 mM) eluent, acidified with trifluoroacetic acid (5.2 mM). The retention time was 5.4 min and the asymmetry factor 0.65. A linear calibration curve from 10 to 1000 μg/ml ( r = 0.99998), with a detection limit of 2.7 μg/ml (25 μl injection volume), a repeatability %RSD of 0.8 (40 μg/ml, n = 5) and reproducibility %RSD of 2.6 were achieved. The proposed method was applied for the determination of carnitine in oral solutions and capsules. No interference from excipients was found and the only pretreatment step required was the appropriate dilution with the mobile phase. Recovery from spiked samples was ranged from 97.7 to 99.7% with a precision (%RSD, n = 3) of 0.01–2.1%.

Determination of carnitine and acylcarnitines in plasma by high‐performance liquid chromatography/electrospray ionization ion trap tandem mass spectrometry

A high-performance liquid chromatography/mass spectrometry method was developed for the determination of carnitine, its biosynthetic precursor butyrobetaine, and eight acylcarnitines in plasma. The procedure includes a solid-phase extraction for carnitine and short- and medium-chain acylcarnitines, and a liquid-liquid extraction for protein-bound long-chain acylcarnitines, followed by separation on a reversed-phase column in the presence of a volatile ion-pairing reagent. Detection was achieved using an ion-trap mass spectrometer run in the tandem mass spectrometry (MS/MS) mode. The choice of the matrix for calibrators, used for quantification of these endogenous compounds, was also investigated. Validation was performed for standard quality controls diluted with 4% bovine serum albumin solution and for spiked plasma quality control samples at concentrations between 0.5 and 80 micromol/L, depending on the compound. Intra- and inter-day precisions for the determination of carnitine were below 3.4% and accuracies were between 95.2 and 109.0%. Application of the method to the diagnosis of pathological acylcarnitine profiles of metabolic disorders in a patient suffering from methylmalonic aciduria is presented. The method allows quantification of carnitine, butyrobetaine, acetylcarnitine and propionylcarnitine, and semiquantitative analysis of medium- and long-chain acylcarnitines. In contrast with other methods, no derivatization step is needed.

Determination of carnitine and acylcarnitines in urine by high-performance liquid chromatography–electrospray ionization ion trap tandem mass spectrometry

A high-performance liquid chromatography–mass spectrometry method has been developed for the simultaneous determination of native carnitine and eight acylcarnitines in urine. The procedure uses a solid-phase extraction on a cation-exchange column and the separation is performed without derivatization within 17 min on a reversed-phase C 8 column in the presence of a volatile ion-pairing reagent. The detector was an ion trap mass spectrometer and quantification was carried out in the MS–MS mode. Validation was done for aqueous standards at ranges between 0.75 and 200 μmol/l, depending on the compound. Carnitine was quantified in urine and comparison with a radioenzymatic assay gave a satisfactory correlation ( R 2=0.981). The assay could be successfully applied to the diagnostic of pathological acylcarnitines profile of metabolic disorders in urines of patients suffering from different organic acidurias.

Chip-Based Quantitative Capillary Electrophoresis/Mass Spectrometry Determination of Drugs in Human Plasma

A chip-based capillary electrophoresis/mass spectrometry (CE/MS) system is described for the on-chip separation and coupled electrospray detection of selected small drug molecule compounds. These studies include the quantitative determination of carnitine and acetylcarnitine in analytical standard solutions as well as imipramine and desipramine in fortified human plasma samples. A clinical human plasma sample was also analyzed following the normal administration of desipramine to a volunteer, and the parent drug was determined using the described chipbased CE/MS technique. In each instance, stable isotope-incorporated internal standards were used. The chip-based CE system was microfabricated from glass and coupled to a micro ion spray device constructed in-house. The atmospheric pressure ionization system employed in this work was a PE Sciex API III tandem triple quadrupole system operated in the selected ion monitoring (SIM) mode. The results from the work reported here demonstrate the feasibility for carrying out rapid (30 s) chipbased quantitative CE/MS determinations of samples containing small-molecule compounds. Using SIM CE/ MS techniques, the described API III quadrupole system provided acceptable ion current electropherograms from subpicomole levels of the targeted compounds loaded onto the chip. The corresponding electropherograms for the standard solution of carnitines at the 1-500 microg/mL level were obtained via SIM CE/MS techniques (R2 > 0.99). In addition, analyses of fortified samples of imipramine desipramine were measured relative to their corresponding d3 internal standards to obtain calibration curves ranging from 5 to 500 microg/mL in human plasma (R2 > 0.99). The intra-assay precision ranged from 4.1 to 7.3% RSD. The intra-assay accuracy ranged from 94.0 to 104%. These results demonstrate the feasibility for on-chip CE separation and electrospray mass spectrometric determination in applications for bioanalytical measurements for these important compounds in synthetic mixtures and human plasma extracts.

Determination of carnitine and acylcarnitines in biological samples by capillary electrophoresis–mass spectrometry

Free carnitine and acylcarnitines (carnitine esters) play an important role in the metabolism of fatty acids. Metabolic disorders can be detected by abnormal levels of these compounds in biological fluids. Capillary electrophoresis–mass spectrometry has the advantage of combining an efficient separation technique with highly selective detection. Therefore, we have developed a method for the determination of carnitine and several of its esters implementing electrospray capillary electrophoresis–mass spectrometry in the positive ion selected reaction monitoring mode. A sheath-flow interface with a mixture of 2-propanol or methanol, water and acetic acid as sheath liquid and nitrogen as nebulizing gas was used. The zwitterionic analytes migrated as cations in the applied electric field using ammonium acetate–acetic acid or formic acid electrolytes. Separations were performed in aqueous, mixed organic–aqueous and non-aqueous media. The influence of the electrolyte composition on the separation efficiency was investigated. The electrospray conditions have been optimized regarding ion current stability and sensitivity. Ammonium acetate (10 mmol/l)–0.8% formic acid in water or 6.4% formic acid in acetonitrile–water (1:1) were used as running buffers for the determination of carnitine and acylcarnitines in human biological samples. Methanol extracts of dried blood spots were analyzed as well as urine and plasma following sample preparation via solid-phase or liquid–liquid extraction. Recoveries approaching 100% were achieved depending on the analytes and sample preparation procedures employed. Endogenous carnitine and acetylcarnitine were determined at concentrations between 2.7 and 108 nmol/ml in normal human urine and plasma. Other acylcarnitines were detected at levels of below the limit of detection to 12 nmol/ml. Good precision (0.8 to 14%) and accuracy (85 to 111%) were obtained; the achieved limits of quantitation (0.1 to 1 nmol/ml) are sufficient to characterize carnitine and acylcarnitine levels occurring as markers for metabolic disorders.

Separation and determination of carnitine and acyl-carnitines by capillary electrophoresis with indirect UV detection

A new capillary zone electrophoretic (CZE) method has been developed for the simultaneous determination of carnitine, acetyl-carnitine, hexanoyl-carnitine, octanoyl-carnitine, decanoyl-carnitine and lauroyl-carnitine with indirect photometric detection. Two selected buffers were evaluated and optimized; the first one used copper sulfate and methanol, and the second used quinine as the principal constituent. Different chemical variables were evaluated and optimized in order to setup the most appropriate system for separating the acyl-carnitines. The robustness test showed that the quantitative responses (areas and heights) were affected mainly by pH and quinine concentration, while the migration times were influenced by quinine concentration, pH, voltage and temperature. The present method achieved times of separation of 20 min for six carnitine compounds with complete resolution of all compounds. The detection limits of the method were between 0.2 and 2.5 μg ml −1 using electrokinetic injection and reproducibility (RSD%) was between 2.1 and 7.7%. The proposed method was applied to determine carnitines in synthetic samples; the recoveries of the compounds were between 95 to 110% in different samples and the errors were lower than 10% in all cases.

Biological Variation of Free and Total Carnitine in Serum of Healthy Subjects

Determination of carnitine (l-β-hydroxy-γ-trimethylaminobutyric acid) in biological samples plays an important role in the diagnosis of diseases with carnitine deficiency (1). In the past few years, and also in this Journal, several spectrophotometric methods for assaying carnitine in serum have been described (2)(3)(4)(5)(6). To our knowledge, however, no data have been published on the biological variation of this serum analyte. For any new test, data on the biological variation generated from the healthy population may be used (a) in setting desirable performance standards or analytical goals, (b) in assessing the utility of conventional population-based reference intervals, and (c) in critically evaluating the significance of changes in serial results from an individual (7). To investigate the analytical and biological variation of free and total carnitine, as well as of the acyl/free carnitine ratio, we collected blood samples once a week for 4 weeks from each of 14 healthy subjects (7 men and 7 nonpregnant women, ages 21–23 years), students of Mathias Belius University. The subjects agreed to maintain current dietary habits, body weight, and exercise program (if any) for the duration of the study. The criteria of the hospital Ethics Committee were respected in this experiment. To minimize sources of preanalytical variation, venous blood specimens were drawn between 0800 and 0900 h after ∼12 h of fasting. The subjects …

Enzymatic determination of carnitine in milk and infant formula

A method has been developed for the routine estimation of carnitine in milk, milk powder and infant formula. Carnitine and its esters were measured following a selective alkaline hydrolysis scheme, with use of the coupled carnitine acetyl transferase-Ellman reaction to form the nitrophenolate anion chromophore. The method was automated using a centrifugal analyser to facilitate high sample capacity and improved precision. Endogenous total carnitine in whole milks varied between 20·2 and 28·1 mg/100 g solids, with free carnitine contributing 55–60% of the carnitine pool. A reliable estimate of the total carnitine content in milk is achieved from the sum of the acid soluble free and short-chain acylcarnitines, with the acid insoluble long-chain esters present at levels below the uncertainty of the methodology. Infant formulas demonstrated more diverse carnitine contents as a consequence of their complex and varied compositions and the increasingly common practice of supplementation.

HPLC determination of carnitine and acylcarnitines in human plasma by means of fluorescence labeling using 2-(4-hydrazinocarbonylphenyl)-4,5-diphenylimidazole.

Carnitine and acylcarnitines are important substances involved in the oxidation and metabolism of fatty acids. An HPLC method is presented for the quantitative analysis of these compounds. The method is based on the detection of fluorescent derivatives of carnitine and short- and medium-chain acylcarnitines labeled with 2-(4-hydrazinocarbonylphenyl)-4,5-diphenylimidazole (HCPI). The labeling of carnitine and acylcarnitines with HCPI was performed at room temperature for 1 h using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as a condensing reagent. The analytes prepared using cation-exchange cartridges were separated on an octadecylsilyl silica gel (ODS) column by a gradient elution system of acetonitrile/Tris-HCI buffer and determined using a synthetic internal standard. Fluorescence detection was performed at 475 nm with excitation at 340 nm. The detection limits for carnitine, acetyl-, propionyl-, hexanoyl- and octanoylcarnitine ranged from 0.24 to 1.97 nmol per ml human plasma, at a signal-to-noise ratio of 3. The within-day and between-day precision of the assay for carnitine and acylcarnitines in plasma samples had relative standard deviations (RSDs) lower than 10.3%. The concentration of free carnitine, acylcarnitines and total carnitine in human plasma could be successfully determined by the proposed method.