Carnitine
(3-hydroxy-4-trimethylamino-butyric acid is metabolite that has a multiple
function, it carries long chain fatty acid for beta oxidation, it is the most
important metabolite for energy production in every cell of the body, this
paper shows the determination of l carnitine in several biological, food and
pharmaceutical samples using different methods of determination such as High
performance ion pair chromatography, Hplc-Uv , capillary electrophoresis,
Capillary electrophoresis-mass spectrometry.

Introduction

acid or ?-hydroxy-gamma-N-trimethylamino-butyrate),
which is sometimes called vitamin BT, is a water soluble amino acid, but it is
not a true vitamin because; its biosynthesized endogenously in liver and kidney
from the dietary amino acid trimethyllysine  (Lheureux & Hantson, 2009).

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 Carnitine is a metabolite which only exists in
eukaryotic cells and has a main functions; indispensable for intermediary
metabolism, to facilitate the transport of fatty acids and acetyl units during
central carbon metabolism between cell compartments (Strijbis et al., n.d.), due to this it is required in mitochondrial oxidation of long
chain fatty acids, in establishing intramitochondrial acyl coenzyme A/ coenzyme
A ratio steady-state (Minkler, Ingalls, & Hoppel,
2005; Strijbis et al., n.d.). the deficiency of primary carnitine can cause a disorder called
autosomal recessive disorder of fatty acid oxidation and It is  caused by a mutation of a gene SLC22A4 which affects
a specific L carnitine transporter called OCTN2, the absence of this
transporter increases the disposal of urinary carnitine  out of the body and its tissue (Guo, Lü, Li, & Wang, 2008). 

In mammalian species carnitine
levels are dependent on diet, because red meat contains high concentrations of
carnitine (containing 500 to 1200 mg/kg), followed by fish, chicken and
milk-derived substances (containing 16 to 64 mg/kg). while in plant sources has
a relatively small concentration(usually b 0.5 mg/kg), but it can be also
synthesized 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 active
form of carnitine whereas the D-carnitine is known to be inactive and it might
cause 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 thus
causing 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 essential
amino 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 to
decreased 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 of
l-carnitine was in chick embryos, which have a decent amount of l-carnitine in
it, but egg itself has none.  When grown
on a carnitine-free synthetic medium, the microorganism Neurospora crassa also
contained carnitine(Koch, Konig, Stangl, & Eder,
2008).

 in the biosynthesis of l-carnitine the methyl
groups come from the essential amino acid methionine, but not from the
macronutrient (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 peptide
linkages undergoes methylation of the e-amino group to yield trimethyllysine
(TML), which is released upon protein degradation. It was also shown that
lysine is converted to carnitine with 6-N-trimethyllysine as an intermediate(Harpaz, 2005; Koch et al., 2008). 

the endogenous production of
l-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 &
Hantson, 2009).  

 L-carnitine it is the most important isomer of
carnitine that has most of the functions; it is one of the most essential
substance that helps to transport fatty acids into the cell matrix thus helps
with the production of ATP and B-oxidation(Mroczkowska, Galla, Na??cz, &
Na??cz, 1997) ,  it is critical
for 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 important
as 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 oxidative
stress, inhibit fatty acid ester accumulation during ischemic events, and
prevent cardiac cell apoptosis(Calò et al., 2006).

 Due the important l-carnitine role of the
oxidation of fatty acid; it can diminish urea cycle enzymes modulation during
transcription by long chain fatty acids thus induces
hyperammonemia 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 between
2 and 12 lmol/kg of body weight/day, that is, from 0.3 to 1.9 mg/kg/day. The FDA has approved the
use of L-carnitine, either taken by mouth or
given intravenously(Sánchez-Hernández,
García-Ruiz, Crego, & Marina, 2010), because many cases of l-carnitine deficiency
have been reported.   Carnitine
deficiency may predispose to chronic fatigue by impairing utilization of long
chain fatty acids in energy metabolism. Long chain fatty acids are the
preferred source of energy by muscle and cardiac cells because they have a high
yield of adenosine triphosphate (ATP), their consumption does not compromise
other cellular functions, and they can be stored in large quantities, Patient with cancer are at risk for carnitine
deficiency due to decreased oral intake and increased renal losses. (Aleisa et al., 2007; Cruciani et
al., 2009, 2012).  Due the importance of L carnitine in
metabolism the determination and detection of l carnitine is significant.

Methods
of determination

High performance ion-pair
chromatography

Pure solid l-carnitine, in the form of oral solution
and capsules was obtained and stored in airtight container, at the same time the
standard stock solution was prepared in water (10,000
microgram/ml) and kept in the refrigerator.  the standard l carnitine solutions they worked
were in the range of (10-1000 microgram/ml) and they were prepared daily in
the mobile phase.  They used a stationary
phase of (C-18) and a mobile phase of octanesulfonate, heptane and
TFA, but since octanesulfonate appeared to get a high peak they used it for
further optimizations.

In sample preparation the oral solution was diluted
using water and an extra dilution was prepared using the mobile phase to get
sample solutions exist among the range of (100-700 microgram/ml).

Whereas the capsules were weighted and dissolved in
water in 10 units with a little shaking of flask, the sample were filtered and
diluted using the mobile phase to reach the range of (100-700 microgram/ml).

The whole chromatographic procedure was done in a room
temperature of (25 ?C) in isocratic mode with a flow
rate (1.2ml/min), filtration was done on the eluent solution before it used.

In the procedure octalosulphate was
added with different concentraitions (0.64, 0.30 and 0.10 mM), it appreard that
the higher concentration of octalosulphate results in the increase of retention
time which is not good for the analysis, so keeping it at the same start up
concentration was better.

 

Fig. 1. Effect of
octanesulfonate concentration in mobile phase on chromatographic
characteristics of l-carnitine (TFA concentration 3.9mM): (A) 0.64mM (tR
l-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 concentration
of TFA in the range of (1.3-5.2mM) appeared to have a diserable results in
lowering the retention time where it was the best at the concentration of 5.3mM

 (Kakou, Megoulas, & Koupparis, 2005)

 

  

Fig. 2. Effect of TFA
concentration 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).

 

High
performance liquid chromatography-uv

In this method l carnitine
was determined in biological samples such as plasma, milk and muscles in cow, In
the HPLC a reversed phased column with fluorescence was used. The mobile phase
contained 30% of acetonitrile with 0.1 ammonium acetate in water and the PH was
controlled by acetic acid and it was (PH 3.5), the separation was performed
with a flow rate of 1.5mL/min.

determination was based on
the synthesis of fluorescent derivative of the carnitines, so the process
required precolumn derivatization by several reagents, the sample was purified
prior the derivatization by solid phase extraction.  a stock solution of l carnitine was prepared
10 mg dissolved in 10 ml of water, several standard solutions were obtained by
the dilution of stock solution with concentrations (0.5, 1, 5, 10 and 50
Microgram/ml).  A cow plasma sample was
obtained and dialyzed, the dialyzing process was used just for removing the
endogenous l carnitine.  Fresh biological
samples of plasma, milk and muscle were collected after 24 g L-carnitine was
given daily for two months to five cows. 
concentrations of L-carnitine in milk and muscle samples were much
higher than in the plasma samples(Cao, Ren, Park, Choi,
& Lee, 2007).

Fig. 3. HPLC chromatograms of L-carnitine in the
biological fluids two months after oral administration of 24 g L-carnitine/day.
Plasma (A), milk (B) and muscle (C).

 

Capillary
electrophoresis

Capillary electrophoresis is known to be an effective
method for the determination of l carnitine and three2 developed method (ITP,
CZE with direct or/and indirect UV detection) was made in comparison with HPLC
method, just to ensure the validation of the method.  The system depends on direct or indirect UV
or 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, carbonate
buffer and acetic acid were prepared in water and stored at a specific
temperature.  The samples were
injected in the CE once with direct uv detection and the other with in direct
detection, in some samples FMOC derivatization was necessary for
electrophoresis 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), CZE
mode with direct UV detection (C); R, response of conductimeter; UV, response
of UV detector; for conditions see in text

Capillary
electrophoresis-Mass spectrometry

In this method l carnitine
samples were obtained from pharmaceutical ingredients ( oral solution, Ampoules
and tablets),  and were diluted with
water at adjusted Ph of (2.5) for a certain concentrations, derivatization was
done to all samples with FMOC after dilution.

Then samples were carried
out in the CE then were injected directly to MS(Sánchez-Hernández et
al., 2010).

 

Fig. 1. MS2
electropherogram, fragmentation pattern and spectra

 

Comparison of the methods

The comparison will be carried out
between the four determination methods from food samples or from eukaryotic
tissues or plasma, all the determination methods aimed to determine and detect
l carnitine in potentially short time, high sensitivity, high concentrations,
with no or less degradation, other limitation and with easiest less complicated
method.  the first method applied was ion
pair chromatography with indirect conductimetric detection with a non-polar C18
and with octanosulphate and TFA eluent 
,in this LC method unlike other methods it has the advantages of no
derivatization step (which implies a low of the procedure), short analysis time
of (5 min), sufficient detection limit (2,7 microgram/ml), systemic peak and
linearity was excellent in all cases, the only thing that requires a special
attention is the fact that TFA is a strong reagent and it should be handled
carefully.

The second method, High performance
liquid chromatography was applied for the determination of l carnitine in
biological samples such as plasma, milk and muscles in cow, this method required
retention time of 4.0 to 4.9 which is potentially low, but since there was a
solid extraction and derivatization, it was time consuming and with a high
cost, the limit of quantitation (LOQ) was unsatis- factory in some cases. This
method demonstrated good specificity, reproducibility, linearity, accuracy and
precision. It could be applied to the detection of L-carnitine concentration in
various biological samples of animals.

The third method, capillary
electrophoresis, three electrophoresis mode were used with indirect or/and
direct Uv detection, all systems modes gave a comparable result, they were
compared to HPLC to ensure the efficiency of the method, the CE analysis was
faster, consumes less solvent and running cost was lower than HPLC, very simple
sample preparation, the indirect uv detection electrophoresis allowed the
determination of L carnitine directly without all the derivatization complex
reactions, while in the mode that depended on direct uv detection
derivatization method was necessary, linearity and sensitivity were good for
all modes,

The last method was capillary
electrophoresis with tandem mass spectrometry, this method was applied for the
determination of l carnitine in pharmaceutical formulation, samples were
diluted in water prior the derivatization with FMOC, the method showed a good
linearity, sensitivity and accuracy, even though this method took a lot of
steps to be done, it ensured the quality control of l carnitine in
pharmaceutical formulation.

 

Conclusion

Regarding the four method that I
mentioned before in my paper, high ion pair chromatography, HPLC, Capillary
electrophoresis and Capillary electrophoresis-mass spectrometry, the best
method for the determination of l carnitine was High performance ion pair
chromatography, because it had a short retention time, with low cost, high sensitivity, and
less complicated method, it didn’t require a pre derivatization of the sample.

capillary electrophoresis – MS was
good for ensuring the quality of l carnitine in pharmaceutical formulation, but
it 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 a
great method with high validation and it was faster comparing to HPLC, yet in
CE direct uv light detection mode derivatization method was necessary and this
mode was unfortunately not applicable. 

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