Carnitine electrophoresis-mass spectrometry. Introduction acid or ?-hydroxy-gamma-N-trimethylamino-butyrate), which

Carnitine(3-hydroxy-4-trimethylamino-butyric acid is metabolite that has a multiplefunction, it carries long chain fatty acid for beta oxidation, it is the mostimportant metabolite for energy production in every cell of the body, thispaper shows the determination of l carnitine in several biological, food andpharmaceutical samples using different methods of determination such as Highperformance ion pair chromatography, Hplc-Uv , capillary electrophoresis,Capillary electrophoresis-mass spectrometry. Introductionacid or ?-hydroxy-gamma-N-trimethylamino-butyrate),which is sometimes called vitamin BT, is a water soluble amino acid, but it isnot a true vitamin because; its biosynthesized endogenously in liver and kidneyfrom the dietary amino acid trimethyllysine  (Lheureux & Hantson, 2009).  Carnitine is a metabolite which only exists ineukaryotic cells and has a main functions; indispensable for intermediarymetabolism, to facilitate the transport of fatty acids and acetyl units duringcentral carbon metabolism between cell compartments (Strijbis et al., n.

d.), due to this it is required in mitochondrial oxidation of longchain fatty acids, in establishing intramitochondrial acyl coenzyme A/ coenzymeA ratio steady-state (Minkler, Ingalls, & Hoppel,2005; Strijbis et al., n.d.

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). the deficiency of primary carnitine can cause a disorder calledautosomal recessive disorder of fatty acid oxidation and It is  caused by a mutation of a gene SLC22A4 which affectsa specific L carnitine transporter called OCTN2, the absence of thistransporter increases the disposal of urinary carnitine  out of the body and its tissue (Guo, Lü, Li, & Wang, 2008).  In mammalian species carnitinelevels are dependent on diet, because red meat contains high concentrations ofcarnitine (containing 500 to 1200 mg/kg), followed by fish, chicken andmilk-derived substances (containing 16 to 64 mg/kg).

while in plant sources hasa relatively small concentration(usually b 0.5 mg/kg), but it can be alsosynthesized in mammalian species as mentioned(Harpaz, 2005; Strijbis et al.,n.d.).

 Carnitine has two isomers  L and D; The L-carnitine known as an activeform of carnitine whereas the D-carnitine is known to be inactive and it mightcause L-carnitine deficiency (Guo et al., 2008), D-carnitine is not only inactive it is also appeared to be toxic  by decreasing the amount of L-carnitine thuscausing a bad effects on human body such as  muscles weakness  and cardiac arrhythmias (Guo et al., 2008).The active form of carnitine; l-carnitine(4-N-trimethylammonium-3-hydroxybutyric acid) is synthesized from the essentialamino acids lysine and methionine (Busquets et al., 2012)with the presence of vitamin C -that’s why the deficiency of ascorbic acid causes a fatigue due todecreased l-carnitine synthesis-,  the iron(Fe2+), vitamin B6 and niacin in the form of nicotinamide adenine dinucleotide(NAD), the first evidence that helped to understand the biosynthesis ofl-carnitine was in chick embryos, which have a decent amount of l-carnitine init, but egg itself has none.  When grownon a carnitine-free synthetic medium, the microorganism Neurospora crassa alsocontained carnitine(Koch, Konig, Stangl, & Eder,2008).

 in the biosynthesis of l-carnitine the methylgroups come from the essential amino acid methionine, but not from themacronutrient (choline) that also has a methyl group, and that g-butyrobetaine(but not gaminobutyric acid or g-dimethylaminobutyrate) is converted to carnitine,Lysine provides the carbon backbone of carnitine. Lysine in protein peptidelinkages undergoes methylation of the e-amino group to yield trimethyllysine(TML), which is released upon protein degradation. It was also shown thatlysine is converted to carnitine with 6-N-trimethyllysine as an intermediate(Harpaz, 2005; Koch et al., 2008).  the endogenous production ofl-carnitine in mammalians is formed in liver, kidneys and the brain, several enzymes are involved in endogenous L-carnitine biosynthesis such as 4-butyrobetaine hydroxylase(Harpaz, 2005; Lheureux , 2009).   L-carnitine it is the most important isomer ofcarnitine that has most of the functions; it is one of the most essentialsubstance that helps to transport fatty acids into the cell matrix thus helpswith the production of ATP and B-oxidation(Mroczkowska, Galla, Na??cz, ??cz, 1997) ,  it is criticalfor energy production in every cell of the body. Without L-carnitine, the normal burning of fat cannot take place(Harpaz, 2005).

 In the heart, L-carnitine becomes very importantas the myocardium prefers to oxidize long-chain fatty acids for energy.  In addition to its role in energy production,L-carnitine helps remove toxins from inside the mitochondria, mediate oxidativestress, inhibit fatty acid ester accumulation during ischemic events, andprevent cardiac cell apoptosis(Calò et al., 2006).  Due the important l-carnitine role of theoxidation of fatty acid; it can diminish urea cycle enzymes modulation duringtranscription by long chain fatty acids thus induceshyperammonemia in higher vertebrates (Harpaz, 2005;Izumi, Izumi, Matsukawa, Funatsu, & Zorumski, 2005).  As a supplement it has been proposed that the daily need for L-Carnitine is between2 and 12 lmol/kg of body weight/day, that is, from 0.3 to 1.9 mg/kg/day.

The FDA has approved theuse of L-carnitine, either taken by mouth orgiven intravenously(Sánchez-Hernández,García-Ruiz, Crego, & Marina, 2010), because many cases of l-carnitine deficiencyhave been reported.   Carnitinedeficiency may predispose to chronic fatigue by impairing utilization of longchain fatty acids in energy metabolism. Long chain fatty acids are thepreferred source of energy by muscle and cardiac cells because they have a highyield of adenosine triphosphate (ATP), their consumption does not compromiseother cellular functions, and they can be stored in large quantities, Patient with cancer are at risk for carnitinedeficiency due to decreased oral intake and increased renal losses. (Aleisa et al., 2007; Cruciani etal., 2009, 2012).

 Due the importance of L carnitine inmetabolism the determination and detection of l carnitine is significant. Methodsof determination High performance ion-pairchromatographyPure solid l-carnitine, in the form of oral solutionand capsules was obtained and stored in airtight container, at the same time thestandard stock solution was prepared in water (10,000microgram/ml) and kept in the refrigerator.  the standard l carnitine solutions they workedwere in the range of (10-1000 microgram/ml) and they were prepared daily inthe mobile phase.  They used a stationaryphase of (C-18) and a mobile phase of octanesulfonate, heptane andTFA, but since octanesulfonate appeared to get a high peak they used it forfurther optimizations. In sample preparation the oral solution was dilutedusing water and an extra dilution was prepared using the mobile phase to getsample solutions exist among the range of (100-700 microgram/ml).

Whereas the capsules were weighted and dissolved inwater in 10 units with a little shaking of flask, the sample were filtered anddiluted using the mobile phase to reach the range of (100-700 microgram/ml). The whole chromatographic procedure was done in a roomtemperature of (25 ?C) in isocratic mode with a flowrate (1.2ml/min), filtration was done on the eluent solution before it used.In the procedure octalosulphate wasadded with different concentraitions (0.64, 0.30 and 0.10 mM), it appreard thatthe higher concentration of octalosulphate results in the increase of retentiontime which is not good for the analysis, so keeping it at the same start upconcentration was better.  Fig.

1. Effect ofoctanesulfonate concentration in mobile phase on chromatographiccharacteristics of l-carnitine (TFA concentration 3.9mM): (A) 0.64mM (tRl-carnitine: 7.

1min, system peak: 3.3min), (B) 0.3mM (tR l-carnitine: 5.7min,system peak: 2.8min) and (C) 0.10mM (tR l-carnitine: 3.5min, system peak:1.

9min).Whereas increasing the concentrationof TFA in the range of (1.3-5.2mM) appeared to have a diserable results inlowering the retention time where it was the best at the concentration of 5.3mM (Kakou, Megoulas, & Koupparis, 2005)      Fig. 2. Effect of TFAconcentration in mobile phase on chromatographic characteristics of l-carnitine(octanesulfonate concentration 0.

64mM): (A) 5.2mM (tR l-carnitine: 5.4min,system peak: 2.7min), (B) 2.6mM (tR l-carnitine: 8.

6min, system peak: 4.3min). Highperformance liquid chromatography-uvIn this method l carnitinewas determined in biological samples such as plasma, milk and muscles in cow, Inthe HPLC a reversed phased column with fluorescence was used. The mobile phasecontained 30% of acetonitrile with 0.1 ammonium acetate in water and the PH wascontrolled by acetic acid and it was (PH 3.5), the separation was performedwith a flow rate of 1.5mL/min.determination was based onthe synthesis of fluorescent derivative of the carnitines, so the processrequired precolumn derivatization by several reagents, the sample was purifiedprior the derivatization by solid phase extraction.

 a stock solution of l carnitine was prepared10 mg dissolved in 10 ml of water, several standard solutions were obtained bythe dilution of stock solution with concentrations (0.5, 1, 5, 10 and 50Microgram/ml).  A cow plasma sample wasobtained and dialyzed, the dialyzing process was used just for removing theendogenous l carnitine.

 Fresh biologicalsamples of plasma, milk and muscle were collected after 24 g L-carnitine wasgiven daily for two months to five cows. concentrations of L-carnitine in milk and muscle samples were muchhigher than in the plasma samples(Cao, Ren, Park, Choi,& Lee, 2007). Fig. 3. HPLC chromatograms of L-carnitine in thebiological fluids two months after oral administration of 24 g L-carnitine/day.Plasma (A), milk (B) and muscle (C). Capillaryelectrophoresis Capillary electrophoresis is known to be an effectivemethod for the determination of l carnitine and three2 developed method (ITP,CZE with direct or/and indirect UV detection) was made in comparison with HPLCmethod, just to ensure the validation of the method.

  The system depends on direct or indirect UVor fluorescence detection, electrochemical and other detectors.  Samples was made from a pure l carnitine sample;stock solution was made from 1mg l carnitine diluted in water and stored at (4?C). fresh stock solution was prepared in acetonitrile, carbonatebuffer and acetic acid were prepared in water and stored at a specifictemperature.  The samples wereinjected in the CE once with direct uv detection and the other with in directdetection, in some samples FMOC derivatization was necessary forelectrophoresis with direct UV detection(Prokorátová, Kvasni?ka, Šev?ík, & Vold?ich, 2005). Fig.

4. Electropherograms of food supplement sample”Chroma Ultra Slim”; ITP mode (A); CZE mode with indirect UV detection (B), CZEmode with direct UV detection (C); R, response of conductimeter; UV, responseof UV detector; for conditions see in textCapillaryelectrophoresis-Mass spectrometry In this method l carnitinesamples were obtained from pharmaceutical ingredients ( oral solution, Ampoulesand tablets),  and were diluted withwater at adjusted Ph of (2.5) for a certain concentrations, derivatization wasdone to all samples with FMOC after dilution.Then samples were carriedout in the CE then were injected directly to MS(Sánchez-Hernández etal., 2010).

 Fig. 1. MS2electropherogram, fragmentation pattern and spectra  Comparison of the methods The comparison will be carried outbetween the four determination methods from food samples or from eukaryotictissues or plasma, all the determination methods aimed to determine and detectl carnitine in potentially short time, high sensitivity, high concentrations,with no or less degradation, other limitation and with easiest less complicatedmethod.  the first method applied was ionpair chromatography with indirect conductimetric detection with a non-polar C18and with octanosulphate and TFA eluent ,in this LC method unlike other methods it has the advantages of noderivatization step (which implies a low of the procedure), short analysis timeof (5 min), sufficient detection limit (2,7 microgram/ml), systemic peak andlinearity was excellent in all cases, the only thing that requires a specialattention is the fact that TFA is a strong reagent and it should be handledcarefully.The second method, High performanceliquid chromatography was applied for the determination of l carnitine inbiological samples such as plasma, milk and muscles in cow, this method requiredretention time of 4.0 to 4.

9 which is potentially low, but since there was asolid extraction and derivatization, it was time consuming and with a highcost, the limit of quantitation (LOQ) was unsatis- factory in some cases. Thismethod demonstrated good specificity, reproducibility, linearity, accuracy andprecision. It could be applied to the detection of L-carnitine concentration invarious biological samples of animals.The third method, capillaryelectrophoresis, three electrophoresis mode were used with indirect or/anddirect Uv detection, all systems modes gave a comparable result, they werecompared to HPLC to ensure the efficiency of the method, the CE analysis wasfaster, consumes less solvent and running cost was lower than HPLC, very simplesample preparation, the indirect uv detection electrophoresis allowed thedetermination of L carnitine directly without all the derivatization complexreactions, while in the mode that depended on direct uv detectionderivatization method was necessary, linearity and sensitivity were good forall modes, The last method was capillaryelectrophoresis with tandem mass spectrometry, this method was applied for thedetermination of l carnitine in pharmaceutical formulation, samples werediluted in water prior the derivatization with FMOC, the method showed a goodlinearity, sensitivity and accuracy, even though this method took a lot ofsteps to be done, it ensured the quality control of l carnitine inpharmaceutical formulation.  Conclusion Regarding the four method that Imentioned before in my paper, high ion pair chromatography, HPLC, Capillaryelectrophoresis and Capillary electrophoresis-mass spectrometry, the bestmethod for the determination of l carnitine was High performance ion pairchromatography, because it had a short retention time, with low cost, high sensitivity, andless complicated method, it didn’t require a pre derivatization of the sample.

capillary electrophoresis – MS wasgood for ensuring the quality of l carnitine in pharmaceutical formulation, butit was with a high cost.HPLC-UV has a low retention time ,but it was expensive due to solid extraction step and derivatization .Capillary electrophoresis was agreat method with high validation and it was faster comparing to HPLC, yet inCE direct uv light detection mode derivatization method was necessary and thismode was unfortunately not applicable. 


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