Research Paper

Selective HPLC Method Development for Soy

Phosphatidylcholine Fatty Acids and Its Mass
Spectrometry
R. D. JANGLE*, R. V. GALGE, V. V. PATIL AND B. N. THORAT
Department of Chemical Engineering, Advanced Drying Laboratory, Institute of Chemical Technology (Formerly UDCT),
N. P. Road, Matunga (E), Mumbai-400 019, India

Jangle, et al.: Phosphatidylcholine Method Development

A novel, efficient and simple approach for soy phosphatidylcholine analysis according to its fatty acid composition
was studied with reverse-phase high-performance liquid chromatography. The reverse-phase high-performance
liquid chromatography analysis was performed isocratically using UV detector and simple mobile phase solvents
consisting of isopropyl alcohol, methanol, and deionized water in the proportion of 70:8:22 v/v. The uniqueness of
the proposed method was the separation of individual fatty acids of soy phosphatidylcholine. The high-performance
liquid chromatography method for soy phosphatidylcholine was validated for linearity with correlation coefficient
of above 0.99 for all the peaks separated according to their fatty acid composition. The intra‑day and the inter‑day
precision studies provided the relative standard deviation of less than 2%. The limit of detection and limit of
quantitation values were also calculated for all the resolved peaks of soy phosphatidylcholine. Also system performance
parameters such as number of theoretical plates, capacity factor, tailing factor, separation factor, and peak resolution
were studied systematically and found well within the acceptable range. The proposed high-performance liquid
chromatography method was successfully applied to soy phosphatidylcholine extracted and purified from deoiled
soy lecithin without any interference of impurities or solvent peaks. Individually, the collected peaks of sample soy
phosphatidylcholine were subjected for mass spectroscopy. The mass spectra showed all the peaks having different
saturated or unsaturated fatty acid chains attached to glyerophosphocholine moiety of soy phosphatidylcholine.
The method developed is economic and well suited for estimation of soy phosphatidylcholine with its fatty acid
composition.

Key words: Phosphatidylcholine, fatty acid, mass spectra, phospholipid, resolution

Phospholipids are major constituents of cell cosmetics domain depends mainly on the PC with
membrane and are found in all tissues and its saturated or unsaturated fatty acid content
subcellular compartments as mixtures of various and in many cases; lecithin with more than 50%
molecular species such as phosphatidylcholine PC content is used. On the other hand, in some
(PC), phosphatidylethanolamine (PE), pharmaceutical formulations, especially those
phosphatidylinositol (PI), sphingomyelin (SM), used for neurological disorders, liver dysfunction
and lysophosphatidylcholine (LPC) depending on and in the preparation of liposome preparations,
the type of polar head groups and the degree of lecithin containing 80-90% PC is desired[5].
unsaturation of the acyl chains [1‑4]. Among these
phospholipids, PC represents a major constituent 1,2-diacyl-sn-glycero-3-phosphocholine (PC) (fig. 1)
of cell membranes. The demand for lecithin with consists of polar head group phosphorylcholine
high PC content from vegetable source is increasing attached to the sn-3 position of glycerol and
these days, particularly in pharmaceutical, cosmetic, differing saturated and unsaturated fatty acids
food and other applications due to their emulsifying esterified to the sn-1 and sn-2 position, whereby,
properties and nonantigenic nature. The application the fatty acid in position sn-1 are preferentially
of vegetable lecithins in pharmaceutical and saturated as a rule[6]. Different fatty acids attached
to glycerol moiety makes PC a very complex
*Address for correspondence molecule and consequently pose difficulty in
E-mail: rdjangle@gmail.com analyzing PC with its exact fatty acid composition.
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MATERIALS AND METHODS

Phosphatidylcholine (≥99% pure) was procured
from Sigma Aldrich, St. Louis, USA. Deoiled
soybean lecithin was procured from Sonic Biochem
Extraction Ltd., Indore, India. The thin layer
chromatographic (TLC) plates and silica gel (100 to
200 mesh size) were obtained from S. D. Fine-Chem
Ltd., Mumbai, India. HPLC grade methanol and
isopropyl alcohol (IPA) were purchased from HiMedia
Fig. 1: Structure of phosphatidylcholine. Laboratories Pvt. Ltd., Mumbai, India. Deionized (DI)
water was prepared by Milli‑Q system (Millipore,
Various analytical techniques are reported such Billerica, MA, USA). All solvents were filtrated
as gas chromatography (GC) [7,8] , thin layer through 0.2 µm Pall Ultipor N66 membrane before
chromatography (TLC) [9,10] , high-performance use. All other reagents used were of analytical grade
thin layer chromatography (HPTLC)[11], and high- and procured from S. D. Fine-Chem Ltd., Mumbai,
performance liquid chromatography (HPLC) [12-16] India.
for lipid analysis. Most of these techniques suffer
from various drawbacks as being time-consuming, Selection and preparation of mobile phase:
insensitive, destructive, or not related to individual Pure PC was dissolved in methanol and injected into
substructures. Now-a-days, techniques such as mass the HPLC system and run in different mobile phase
spectrometry (MS) coupled with GC or HPLC are systems. Various proportions of mobile phase were
used extensively for better resolution[17-21]. The use tried using isopropyl alcohol, methanol, and water.
of highly sophisticated NMR technique is well It was found that isopropyl alcohol, DI water and
suited to quantify phospholipid analogs and needs methanol in the proportion of 70:22:8 v/v has given
less sample preparation but still need to be coupled satisfactory results as compared to the other mobile
with MS for better molecular analysis[6,22-25]. HPLC phases. The isocratic elution was carried out with the
offers the separation of lipid classes using the flow rate of 0.5 ml/min at 25° and a wavelength of
205 nm was used for detection.
normal phase mode and additionally the separation
according to the different fatty acid residues of an
Instrument details and optimization:
individual lipid in the reversed phase mode. There
HPLC system used was (Agilent Technologies,
are no reports in literature, which showed separation
India 1200 Series) equipped with ChemStation
of PtdCho with its varying fatty acid composition
software, a model G1322A degasser, a model
using isocratic reverse-phase HPLC method with
G1311A quaternary pump, auto sampler, a model
UV detector. G1316A column oven and variable wavelength
UV/Vis detector. The separation was performed on
This paper presents an efficient, economic RP‑HPLC Zorbax Eclipse XDB‑C18 column (150×4 mm 2, 5
setup to separate PC by their fatty acid composition. μm).
In this investigation attempts were made to
find out various fatty acid homologs present in Any solvent remaining in the system from the
phosphatidylcholine. In this respect, the individual previous analysis was washed out by passing
peaks were analyzed using mass spectroscopy in methanol:water (50:50) for 10 min at 1 ml/min
the positive and negative ion mode. From several flow rate with purge valve open. After closing the
experiments and attempts, the possible fatty acid purge valve, 100% methanol was passed for 30 min
present in PC can be predicted, however, the position through column for proper column washing. After this
of a particular acid group and that of a double bond step, for column conditioning and base line stability,
in PC is very difficult to predict by this method. Not isopropyl alcohol, DI water and methanol in the
withstanding these drawbacks, however, the developed proportion of 70:22:8 v/v mobile phase was passed
method is still a useful technique for discerning through HPLC system for 1 h at 1 ml/min flow rate.
various homologues of PC. Injection volume was set to 10 µl.
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Standard solution preparation: limit of detection (LOD), and limit of quantitation

Standard PC 50 mg was accurately weighed and (LOQ)[26].
transferred to a 50 ml volumetric flask and the
volume was made with HPLC grade methanol. Linearity:
Solutions of 100, 200, 400, 600, 800, and 1000 μg/ml The linearity of measurement was evaluated by
were made by transferring the aliquot from stock analyzing different concentrations (100 to 1000 μg/ml)
solution and the volume was made with methanol in of the standard solutions. Calibration curve was
each case. Further standard solutions were prepared constructed for combining all the six peaks as well
freshly each day by appropriate dilution of stock as individual peaks showed in Table 1 by plotting
solution with methanol for intraday as well as average peak area against concentration of sample and
interday analysis. regression equation was computed. All the samples
were analyzed in triplicate.
Preparation of sample from deoiled lecithin:
Deoiled soy lecithin powder was extracted with Precision:
95% ethanol in the ratio of 1:7 w/v in a 500 ml The precision of the method was investigated with
capacity stir tank reactor with overhead stirrer respect to repeatability and intermediate precision.
arrangement. The extraction was carried out in The repeatability (intra‑day precision) of the method
air tight reactor with constant stirring at 40° for was evaluated by assaying three replicate injections
2 h with 170 rpm. After stirring, the mixture was of the PC standard at concentrations of 200, 400
filtered and the filtrate was dried in a rotary vacuum and 600 μg/ml on the same day at different times.
evaporator (Buchi Model RII, Switzerland) at The percentage relative standard deviation (% RSD)
40°. Further, the dried sticky mass of PC rich of the peak area was calculated by combining
fraction was purified using silica gel column all the peaks. Intermediate precision (inter‑day
chromatography and methanol fraction containing precision) was demonstrated by evaluating the
purified PC (previously optimized) was collected relative peak area at three different concentration
and methanol from the fraction was evaporated levels as taken in intra‑day study that cover the
completely to get purified PC in sticky form. assay method. The precision was expressed as
For analysis, weighed 10 mg of purified PC in % RSD of the system and was calculated from
volumetric flask and volume made to 10 ml by the combined peak area mean values. The
dissolving in HPLC grade methanol (1 mg/ml). Then intra‑day (n=3) and inter‑day (n=3) % RSD are
this sample was analyzed by proposed RP‑HPLC given in Table 2.
method for its assay purity.
Accuracy study:
Mass spectrometry of PC sample: The accuracy of the method was tested by
Purified PC fraction collected from silica column performing recovery studies at three levels of PC
was analyzed by proposed RP‑HPLC method and reference standard added to the samples. Three
carefully collected the elutions of major four peaks different volumes (0.5, 1, and 1.5 ml) of the
of purified PC fractions isolated from HPLC column standard solution (containing 100 μg/ml of PC in
in different vials and analyzed separately using mass methanol) were added to the sample solution of
spectrometry and subjected to fragmentation of fixed concentration (132.5 μg/ml) and analyzed
individual peaks by MS‑MS. Mass spectrometer used by the proposed HPLC method. All the samples
was of 6420 Series Triple Quadrupole LC/MS, Agilent TABLE 1: VALIDATION PARAMETERS
Technologies (USA), model 410 Prostar Binary LC Standard RT Correlation Slope LOD LOQ
with 500 MS IT PDA detectors with direct Infusion PC peak (Min) coefficient (R2) (µg/ml) (µg/ml)
Mass with ESI. 1 10.92 0.995 0.159 7.26 22
2 12.751 0.996 1.896 2.18 6.61
3 14.965 0.997 6.751 1.92 5.82
Validation of the method:
4 16.864 0.997 1.885 2.48 7.53
Validation of the analytical method was done 5 18.077 0.995 1.101 2.89 8.76
according to the International Conference 6 23.278 0.991 0.493 3.06 9.29
on Harmonization guidelines. The method was Combined – 0.998 12.382 9.36 28.36
validated for linearity, precision, accuracy, LOD=Limit of detection, LOQ=Limit of quantitation

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TABLE 2: PRECISION STUDY where, W=width at 5% of the peak height, f=distance

Concentration (µg/ml) Intra‑day Inter‑day between maximum and the leading edge of the peak.
Area % RSD Day Area % RSD
200 2341.6 0.477 1 2341.6 0.714
The separation factor (α) describes the relative
2331.2 2 2375.2
2319.4 3 2360.8
position of the two adjacent peaks. The separation
400 4824.7 0.283 1 4824.7 0.403 factor or relative retention was calculated using the
4845.9 2 4830.1 capacity factor (k) as peaks’ separation depends
4850.3 3 4860.9 on the components’ interaction with the stationary
600 7579.0 0.294 1 7579 0.254 phase. The separation factor can be calculated by
7545.3 2 7601.2
the Eqn., [27], α=(k B/k A)….(5), where, k A=capacity
7587.4 3 7617.5
N=3, RSD=Relative standard deviation
factor of the 1 st peak, k B =capacity factor of the
2nd peak.
were determined in triplicate. The % recovery
Peak resolution (R) is not only a measure of the
was calculated by using the following formula, %
separation between two peaks, but also the efficiency
Recovery=[(b‑a)/c]×100…(1), where, a – amount of
of the column. It is expressed as the ratio of the
PC found in the sample before addition of standard
distance between the two peak maxima. R=[2(t2‑t1)/
PC, b – amount of PC found after addition of
(W 1 +W 2 )]…(6), where, t 1 =retention time of the
standard PC, c – amount of standard PC added.
1st peak, t1=retention time of the 2nd peak, W1=1st peak
Limit of detection and limit of quantitation: width at base, W2=2nd peak width at base.
The limit of detection (LOD) and limit of
quantification (LOQ) of the developed method RESULTS AND DISCUSSION
were determined by injecting progressively low
concentrations of the standard solutions using the The HPLC separation of soy PtdCho was carried
developed RP‑HPLC method. The LOD is the out on C18 column by an isocratic elution with
smallest concentration of the analyte that gives a isopropyl alcohol, DI water, and methanol in the
measurable response (signal‑to‑noise ratio of 3). proportion of 70:22:8 v/v. The flow rate was constant
The LOQ is the smallest concentration of the at 0.5 ml/min and the column temperature was at
analyte, which gives response that can be accurately room temperature (25±1°). The UV wavelength
quantified (signal‑to‑noise ratio of 10). was set at 205 nm. There was no interference from
impurities present in the PC sample as observed at
System performance parameters: the detection wavelength. The sharp and symmetrical
The number of theoretical plates (n) measures the peaks of PC were obtained when analyzed under these
sharpness of the peaks and therefore the efficiency conditions.
of the column. The number of theoretical plates was
calculated according to US pharmacopeia[27] where, The chromatogram of standard PC peaks is as shown
the peak width at the base was used for calculation. in fig. 2a. Phosphatidylcholine is very complex
n=16×(t/Wb)2 …(2), where, Wb=peak width at base, molecule containing glycerol, fatty acids, phosphate
t=retention time of peak. and choline groups. Pure PC contains constant
choline and phosphate group with various fatty acids
The capacity factor (k’) gives an indication attached to its glycerol backbone. The proposed
of how long each component is retained on the RP‑HPLC method was developed in such a way
column (i.e., how long the component is retarded that it gives various peaks of PC having different
by the stationary phase than it spends in the mobile fatty acid chains at different retention times.
phase). The capacity factor was calculated by the Thereafter, all the PC peaks were considered for
Eqn., k’=(t R ‑t M )/t M ….(3), where, t M =unretained validation parameters study and the standard curve
peak’s retention time, tR=retention time of the peak was prepared separately for individual peaks as it
of interest. was related to their separation from each other with
better resolution. The retention times for various PC
The tailing factor (T) measures the asymmetry of the peaks were found to be 10.92, 12.75, 14.97, 16.86,
peak and is calculated using the Eqn., T=(W/2f)….(4), 18.07, and 23.28 min.
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The major four fractions of elution from HPLC graph 780 (M+23). The fatty acid group giving molecular
were individually collected and subjected to mass weight of 780 may consist of 16 carbon atoms or
spectrometry in order to find the type of fatty acid 18 carbon atoms having 3° of unsaturation (double
groups present in the extracted and purified PC from bond). The double bonds present in this core can be
deoiled soy lecithin. The major peaks representing the associated with one fatty acid or it can be associated
retention time of 12.35, 15.57, 17.44, and 18.63 were with two fatty acids. The peak corresponding to the
collected. The mass spectra of all four peaks and its retention time 15.57 has a mass of 782 (M+23) which
fragmentation patterns are as shown in fig. 3. The may correspond to one fatty acid with 16 number
molecular weight of the peak corresponding to the of carbon atoms and one with 18 number of carbon
retention time of 12.35 was found to have a mass of atoms with two double bond present on either of the
fatty acid chain or one double bond on each fatty acid.
The molecular weight of the peak corresponding to the
retention time of 17.44 has a mass of 758 (M+23).
The molecular weight 758 is associated with two fatty
acids of 16 carbon number without unsaturation. The
molecular weight of the peak corresponding to the
retention time 18.63 was found to be 784 (M+23),
which may contain fatty acid with 16 number of
carbon atoms and one with 18 number of carbon
atoms with one double bond on either of the fatty
acid. The major fragmentation peak at 184 corresponds
a
to phosphocholine. The reason behind quoting of 16
and 18 carbon atoms is due to the fact that the soya
lecithin is known to contain majorly oleic, palmitic,
palmitoleic, linoleic, α‑linolenic and stearic acid, as
per the standard literature. PC from natural source is
a very complex molecule and mainly contain various
fatty acids. The fatty acid side chain present in PC
may vary with the source of its origin, and also
possibility of extensively peroxidized samples can
leave a highly saturated PC residue.
b
Fig. 2: RP‑HPLC chromatograms of phosphatidylcholine.
RP‑HPLC chromatogram of standard phosphatidylcholine (a) and
The method was validated for linearity, precision,
extracted phosphatidylcholine (b). accuracy, LOD, and LOQ. PC has shown good

Fig. 3: Mass spectra of phosphatidylcholine sample peaks.

Mass spectra of sample PC peaks with retention time 12.35 (a), 15.57 (b), 17.44 (c) and 18.63 (d).

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TABLE 3: RECOVERY STUDIES

Analyte Conc of PC before addition Std PC added Conc of PC after addition % Recovery % Mean % RSD
(µg/ml) (a) (µg/ml) (c) (µg/ml) (b)
132.5 100 231.6 99.1
132.5 100 230.1 97.6
132.5 100 231.7 99.2
132.5 200 331.5 99.5
PC 132.5 200 332.1 99.8 99.26 0.714
132.5 200 330.8 99.15
132.5 300 430.2 99.23
132.5 300 431.5 99.67
132.5 300 432.8 100.1
N=3, RSD=Relative standard deviation, PC= Phosphotidylcholine

linearity in the range of 100-1000 µg/ml for TABLE 4: SYSTEM PERFORMANCE PARAMETERS OF
individual peaks (Table 1) as this was the working DEVELOPED RP HPLC METHOD OF PC
Factor Peak
concentration range for the sample analysis.
1 2 3 4 5 6
The intra‑day and inter‑day precisions of PC No of theoretical Plates (n) 11253 13498 12651 15404 12797 16145
are presented in Table 2. These results show the (Acceptable limits, n>2000)
acceptable precision of the method, with % RSD Capacity factor (k’) 1.77 2.25 2.82 3.32 3.63 5.01
(Acceptable limits,
values much lower than 2%. The result also shows
10>k’>2)
that an excellent correlation exists between the Tailing factor (T) 1.164 1.167 1.162 1.138 1.086 1.243
peak area and the concentration of drugs within the (Acceptable limits T≤2)
concentration range. The accuracy of the method was Separation factor (α) 1.27 1.25 1.18 1.09 1.38
(Acceptable limits α > 1)
evaluated by adding the standard solution of 100,
200, and 300 (µg/ml) to the known concentration Peak resolution (R) 3.76 3.8 3.06 1.74 6.12
(Acceptable limits R>2)
of sample solution. The recovery at three different
levels of PC concentrations was obtained with an
average of 99.26% (Table 3) which was well within impurities present in the lecithin as showed in fig. 2b.
the limit of 98 to 102%. The LOD and LOQ for The results showed 99% purity of the purified PC.
smallest PC peak was found to be 7.26 and 22 μg/ml, Thus, it can be said that the proposed method of
respectively (Table 1), which also signifies the high RP‑HPLC was very much suitable for quantification
sensitivity of this method based on the signal‑to‑noise and analysis of PC.
ratio for LOD and LOQ. The signal‑to‑noise ratios
were 6.18 and 20.39, respectively. A novel, convenient, rapid, accurate, and precise
RP‑HPLC method was developed for the estimation
Once a method or a new system is proposed, it is of PC with various fatty acid chains from soybean
important to check the suitability of the method/ source. The assay provides a linear response across a
system against certain set parameters. The parameters wide range of concentrations. The low intra‑day and
such as number of theoretical plates, capacity factor, inter‑day with % RSD as low as 2, gives excellent
tailing factor, separation factor, and peak resolution recoveries of the eluted moieties. The proposed
allow the comparison of the peak shape, its peak method assures prolong life of column and system
and the baseline resolution. Alternatively, these due to simple organic solvents used in the mobile
parameters can be calculated experimentally to phase. The proposed method has been developed
provide excellent quantitative method of performance using UV detector which has made the method more
analysis (Table 4). economic than other methods reported in literature.
The mass spectrometry results confirmed that
PC extracted from crude deoiled lecithin by solid– various PC peaks from HPLC chromatogram were
liquid and liquid–liquid extraction followed by of different fatty acid chains. PC with its various
gradient column chromatographic separation as fatty acid chains can be separated by preparative
mentioned in the literature [28]. This extracted and chromatography in future using the proposed method
purified PC was successfully analyzed by the in economic way. Also, the proposed RP‑HPLC
proposed HPLC method with no interference of other method was successfully worked for extracted and
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