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Determination of Phospholipids in Milk by HPLC
with Evaporative Light Scattering Detector: Optimization and Validation
Ferreiro T, Gayoso L and Rodríguez-Otero JL*
Universidade de Santiago de Compostela, Spain
Submission: February 13, 2017; Published:March 03, 2017
*Corresponding author: Rodríguez-Otero JL, Instituto de Investigación e Análises Alimentarias, Universidade de Santiago de Compostela, Facultade de Veterinaria, 27002 Lugo, Spain, Tel:+34 982822408; Email: firstname.lastname@example.org
How to cite this article: Ferreiro T, Gayoso L, Rodríguez-Otero J. Determination of Phospholipids in Milk by HPLC with Evaporative Light Scattering Detector: Optimization and Validation. Dairy and Vet Sci J. 2017; 1(3): 555562. DOI: 10.19080/JDVS.2017.01.555562
Phospholipids are part of the milk fat globule membrane. Functional and technological properties make them interesting for the development of functional foods or as ingredients of technological interest. In this study an analytical procedure for determination of major phospholipids in milk (phosphatidyl Ethanolamine, phosphatidylinositol, phosphatidyl Choline, phosphatidyl Serine and sphingomyelin) by HPLC with evaporative light scattering detector was optimized and validated. The best calibrations were achieved when an exponential model was applied; the squared correlation coefficients show a satisfactory linearity ranging from 0.975 to 0.993. The limit of detection for the different phospholipids ranged from 0.17μg for phosphatidyl Serine to 0.61μg for phosphatidyl Ethanolamine; and the limit of quantification from 0.40μg for phosphatidyl Serine to 1.26μg for phosphatidyl Ethanolamine. The intra-day and inter-day precision of the method was below 10% for all the compounds except phosphatidylinositol in the intra-day assay with a value of 12.4%. However, the values of intra-day and inter-day repeatability show that the variations, due to the extraction procedure, do not depend whether the assays were performed after short intervals of time or not. The recovery ranged from 74% for phosphatidil Serine to 112% for phosphatidilcoline. The addition of formic acid to the mobile phase in order to achieve an acidic buffer extends the column life considerably.
Phospholipids are present in all living organisms and are the main structural and functional compounds of cellular membranes. They are amphipathic molecules with a hydrophobic moiety and a hydrophilic head group. The glycerophospholipids are characterized by a diglyceride that is covalently bonded to a phosphate group by an ester linkage, and different organic groups (choline, serine, ethanolamine and inositol) may be bound to the phosphate group. The sphingophospholipids consist of a sphingoid base on which a fatty acid is bound to form a ceramide and on this ceramide unit is linked an organophosphate group.
Phospholipids are recently been taken more into consideration because of their nutritional and technological characteristics . Their inhibitory effect on some types of cancer [2-5], their ability to reduce blood cholesterol levels [5,6] and enhance brain functioning [5,7], their anti-bacterial
and anti-inflammatory activity [5,8] and their protective effecton gastric mucosa  have been studied. Additionally, their emulsifying properties can be used in several applications in the food, pharmaceutical and cosmetic industry .
High-performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD) is a useful method for the determination of phospholipids in food matrices . Most of the published HPLC-ELSD methods are normal-phase, with a silica gel or chemically modified silica gels (diol, cyanopropyl, aminopropyl phases) stationary phase and a gradient or isocratic elution, with different solvents; particularly, chloroform: methanol: ammonium hydroxide [12,13] and hexane: isopropanol: water/acids/bases . Recently Pimentel et al.  reviewed the analysis procedures of phospholipids in dairy products including sample extraction and analysis by GC or HPLC using ELSD, CAD and MS detectors.
The aim of this work was to optimize a HPLC-ELSD method
for qualitative and quantitative determination of the major
phospholipids present in milk: phosphatidyl Ethanolamine
(PE), phosphatidylinositol (PI), phosphatidyl Choline (PC),
phosphatidyl Serine (PS) and Sphingo Myelin (SM).
For sample extraction, all the reagents were of analytical
grade, chloroform and methanol from Sigma-Aldrich (St. Louis,
USA), HCl (25%) and NaCl from Panreac (Barcelona, Spain). For
HPLC analysis, HPLC-grade chloroform, HPLC-grade methanol
and ammonium hidroxide (max. 33%) were supplied by Sigma-
Aldrich. Formic acid (98%) and HPLC-grade water were from
Phospholipids standards PE and PC (from bovine brain) and
PI (from bovine heart) were obtained from Larodan (Malmö,
Sweden) and PS and SM (from bovine brain) from Sigma-Aldrich.
The extraction procedure was a modification of the method
described by Rombaut et al. : 5 g of quark or 10 g of milk
were mixed with 20mL of distilled water. The mixture was
transferred into a separatory funnel and 80mL of chloroform:
methanol (2:1vol/vol) was added. After shaking for 2 minutes,
the mixture was separated into two layers, and the lower
chloroform layer was released. Sometimes, it was necessary
to centrifuge (318 x G, for 15-30 minutes at 10 °C to break the
emulsion. Then, 40mL of chloroform: methanol (20:1vol/vol)
was added to the upper phase. This step was repeated and the
two chloroform phases were separated. In a fourth step, 40mL of
chloroform: methanol: water with 1M HCl and 0.9% (w/v) NaCl
(86:14:1 vol/vol/vol) was used and the lower phase was pooled
with the other three, and then evaporated using a rotary vacuum
evaporator at 35ºC. The lipid sample was redissolved in 10mL
of chloroform: methanol (88:12vol/vol), filtered with a syringe
filter (15mm×0.20μm), transferred into an amber vial and stored
at -42°C until HPLC analyses. All samples were extracted and
injected in duplicate.
Phospholipid separations were carried out using a Shimadzu
HPLC system (Kyoto, Japan) composed of the following units:
degasser, solvent delivery module, controller module, column
oven, Rheodyne (Shimadzu) manual injector and an interfacemodule. The detector was a Shimadzu ELSD-LTII. The optimal
parameters stabilised for the detector were: pressure of gas
nebulizer (N2) 3.5 bar, temperature 50 °C and gain 3. A Prevail
silica column (150×3mm) with 3 μm particle diameter (Grace
Davison, Deerfield, IL, USA) and a precolumn with the same
packing was used.
The gradient elution was a linear gradient of chloroform:
methanol: buffer (0.5% formic acid with ammonium hydroxide
until pH 6) 80:19.5:0.5 (vol/vol/vol) at t=0 minutes to 60:33:7
(vol/vol/vol) at t=17 minutes. The initial conditions were
restored at t=20 minutes and the time required to reequilibrate
the column was 15 minutes. The flow rate of the mobile phase
was 0.5mL/minute, the temperature of the column oven was
35°C and the injection volume was 20μL.
In the first stage of optimization of the chromatographic
conditions, a mobile phase gradient of chloroform, methanol and
1M formic acid buffer, adjusted to pH 3 with triethylamine, as
described by Rombaut et al.  was used. By this procedure
the separation of the five major phospholipids (PE, PI, PS, PC and
SM) was achieved. However, the resolution between PS and PI
peaks was not acceptable. Therefore, several modifications were
made with the aim of achieving a total separation.
Since the addition of organic ions to the mobile phase
improves the resolution of acidic phospholipids such as PS and
PI , pH and buffer concentration were changed. Buffers of
different pH (3.5 and 4.0) and at different concentrations (1M
and 2M formic acid) were tested, but without changing the
gradient described in the method .
By the buffer modification the PS and PI separation was not
improved. Therefore, it was decided to vary the gradient and
maintain the initial buffer (1M formic acid, pH 3)
Finally, the conditions that achieved the separation of all
Time 30 min: mobile phase returns to initial conditions.
Under these conditions an acceptable separation was
achieved; but a prolonged use of triethylamine caused significant
fluctuations in the baseline and ghost peaks appeared after
minute 15, preventing the quantification of the phospholipids.
According to other authors  another drawback of the use
of modifiers like triethylamine and formic acid is the gradual
deterioration in the PS peak shape.
After that, tests were performed without the use of buffer
(chloroform: methanol) and substituting the buffer by water
(chloroform: methanol: water). In both cases, as expected, the
separation of all peaks was not achieved, since triethylamine and
formic acid increase the ELSD response .
Then it was decided to seek an alternative to triethylamine.
Before testing mobile phase mixtures in gradient, a test in
isocratic with isopropanol, hexane and water was made, but the
results were not satisfactory.
Subsequently, different gradients of chloroform: methanol:
ammonia, mobile phases used by other authors for the separation
of phospholipids [13,21] were tested, and the separation of
PE, PI, PS, PC and SM was acceptable. But the use of ammonia
has a major drawback, since its basic pH dissolves silica and
the column life diminishes . To solve this problem, formic
acid was added to the solution of ammonia in order to achieve
an acid pH. Different mixtures of formic acid and ammonia,
and different gradients were tested to optimize the separation
conditions, for the identification and quantification of the five
major phospholipids present in milk. The details of the final
chromatographic method were described in section 2.5.
The resolution of the peaks was 4.5 for PE-PI, 4.7 for PIPC,
2.7 for PC-PS and 1.2 for PS-SM. In all cases the resolution
is higher than 1.5, except between the peaks of PS and SM. A
resolution of 1.5 indicates the complete separation of the two
components; whereas with a resolution of 1.0 the non-separated
area is about 4% .
Each peak matches with a type of phospholipid, itself made
up of molecular species with different fatty acids. This fact may
explain the existence of peaks with shoulders and also double
peaks. For the specific case of SM, when a standard of bovine
origin was analyzed separately from the other phospholipids, 3
sub-peaks were detected, while in combination with the other
phospholipids, it was observed that SM eluted as 2 subpeaks
(Figure 1). Double or triple peak of the SM has been described
in several studies [19, 23-25]; this has been attributed to the
presence or absence of an extra hydroxyl group or due to the
different fatty acids or sphingoid groups.
In relation to the light scattering detector parameters, the
gain can be modified from 1 to 12, and it is important to choose
a gain that does not saturate the detector. The nitrogen pressure
should be fixed at an optimum flow of gas to produce an adequate
signal to noise ratio. The evaporating temperature is another
important factor; at higher temperature, the baseline shows
more noise; but the choice temperature has to be enough to
evaporate the mobile phase, avoiding the analyte volatilization.
Different temperatures (50 °C, 55 °C, 60 °C, 65 °C and 80 °C) were
tested and it was noted that when temperature increases, the
response of SM decreases. According to other authors  this
could be due to the evaporation of some free fatty acids of low
boiling point. These values were finally established as follows:
gain 3, nitrogen pressure 3.5 bar and evaporation temperature
After the chromatographic conditions and the detection
parameters were established, the method validation was carried
To evaluate linearity of the ELSD, two different equations
were applied: linear and exponential. The response of the ELSD
has been described as linear [13,17] and non-linear [26,27].
In the present study, the best results were obtained with the
exponential model, which was plotted on a logarithmic scale to
obtain a linear calibration. The calibration curves were calculated
by applying logarithms to the area values and the mass of lipid
standard injected on column. Six levels of concentration were
used for the calibration of each compound. The calibration
equations, correlation coefficients (R2) and concentration range
for PE, PI, PS, PC and SM are shown in Figure 2. The R2 values
show a satisfactory linearity, ranging from 0.993 to 0.975.
The limits of detection (LOD) and quantification (LOQ) were
determined as the amount injected that provided a signal to
noise ratio of 3 and 10 respectively. The LOD for the different
phospholipids ranged from 0.17 μg for PS to 0.61 μg for PE and
the LOQ from 0.40 μg for PS and 1.26 μg for PE (Table 1). Similar
values were previously reported [19,28].
For the determination of intra-day precision, the same
sample (raw milk) was extracted 5 times, and each extraction
was injected in duplicate on the same day. To evaluate the interday
precision, 5 extractions were performed on 5 different days
and each injected twice.
For all the compounds, the RSD% was below 10%, except for
PI in the intra-day assay with a RSD% of 12.4, and there is not a
higher tendency for RSD% in inter-day assays (Table 2). These
results show that the variations due to the extraction procedure
are quite constant and they do not depend on the time factor. The
RSD% obtained was in accordance with the results reported by
other authors [13,14].
To establish the efficiency of the extraction procedure, a
mixture of the five phospholipids was added to the milk sample.
The results range from 74% for PS to 112% for PC (Table 3).
Taking into account the complexity of the extraction process, the
results are acceptable.
The HPLC-ELSD is an adequate technique for the analysis of
the main phospholipids present in milk. The best calibrations
were achieved when an exponential model was applied. The
square correlation coefficients, limits of detection, limits of
quantification, and intra-day, inter-day precision and recoveries
were acceptable. However, the values of intra-day and inter-day
repeatability show that the variations, due to the extraction
procedure, do not depend whether the assays were performed
after short intervals of time or not.
Since the basic pH of the ammonia used in the mobile phase
dissolves silica, the addition of formic acid to the mobile phase
in order to achieve an acidic buffer, extends the column life