U.S. patent application number 15/317441 was filed with the patent office on 2017-04-13 for process for the purification of l-alpha-glycerophosphorylcholine.
The applicant listed for this patent is Chemi S.P.A.. Invention is credited to Mauro Anibaldi, Fabrizio Cocchi, Lorenzo De Ferra, Maurizio Zenoni.
Application Number | 20170101425 15/317441 |
Document ID | / |
Family ID | 51230038 |
Filed Date | 2017-04-13 |
United States Patent
Application |
20170101425 |
Kind Code |
A1 |
De Ferra; Lorenzo ; et
al. |
April 13, 2017 |
PROCESS FOR THE PURIFICATION OF
L-ALPHA-GLYCEROPHOSPHORYLCHOLINE
Abstract
A process for the purification of
L-.alpha.-glycerophosphorylcholine is described, wherein
L-.alpha.-glycerophosphorylcholine is crystallized from DMSO or
from a mixture of DMSO with at least another solvent, preferably
selected from water, alcohol, halogenated solvents, ethers, esters
and/or amides. Such a process allows to obtain
L-.alpha.-glycerophosphorylcholine having a purity greater than
99.5%, preferably greater than 99.7%, even more preferably greater
than or equal to 99.9%. A method for determining the purity of
L-.alpha.-glycerophosphorylcholine is also described, comprising
the elution of L-.alpha.-glycerophosphorylcholine through an HPLC
column having an amino stationary phase, and subsequent detection
of L-.alpha.-glycerophosphorylcholine itself, and any impurity
thereof, by means of an Evaporative Light Scattering Detector
type.
Inventors: |
De Ferra; Lorenzo; (Roma,
IT) ; Anibaldi; Mauro; (Monte Porzio Catone (RM),
IT) ; Zenoni; Maurizio; (Ferentino (FR), IT) ;
Cocchi; Fabrizio; (Roma, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chemi S.P.A. |
Cinisello Balsamo |
|
IT |
|
|
Family ID: |
51230038 |
Appl. No.: |
15/317441 |
Filed: |
June 9, 2015 |
PCT Filed: |
June 9, 2015 |
PCT NO: |
PCT/IB2015/054346 |
371 Date: |
December 9, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07F 9/106 20130101;
B01D 15/08 20130101; G01N 30/02 20130101; G01N 2030/027 20130101;
G01N 21/49 20130101; C07F 9/091 20130101; C07F 9/103 20130101; C07F
9/65742 20130101 |
International
Class: |
C07F 9/10 20060101
C07F009/10; G01N 21/49 20060101 G01N021/49; G01N 30/02 20060101
G01N030/02; B01D 15/08 20060101 B01D015/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 10, 2014 |
IT |
MI2014A001053 |
Claims
1. A process for the purification of
L-.alpha.-glycerophosphorylcholine, wherein
L-.alpha.-glycerophosphorylcholine is crystallized from DMSO or a
mixture of DMSO with at least another solvent.
2. The process according to claim 1, wherein said at least another
solvent is selected from water, alcohol, halogenated solvents,
ethers, esters and/or amides.
3. The process according to claim 1, wherein said alcohol is
methanol, ethanol, isopropanol and/or n-butanol; said halogenated
solvent is methylene chloride; said ether is tetrahydrofuran; said
ester is ethyl acetate; said amide is DMF.
4. The process according to claim 1, wherein DMSO is used in an
amount comprised between 2 and 100 parts by volume per part by
weight of L-.alpha.-glycerophosphorylcholine.
5. The process according to claim 4, wherein said at least another
solvent is used in amounts comprised between 0.01 and 10 volumes
per volume of DMSO.
6. The process according to claim 1, wherein said crystallization
is carried out at a temperature ranging between 0.degree. C. and
100.degree. C.
7. L-.alpha.-glycerophosphorylcholine having a purity greater than
99.5%.
8. L-.alpha.-glycerophosphorylcholine having a content of its
.beta. isomer and/or of cGP lower than 0.1%.
9. A method for determining the purity of
L-.alpha.-glycerophosphorylcholine comprising eluting
L-.alpha.-glycerophosphorylcholine through an HPLC column having an
amino stationary phase, and subsequent detecting
L-.alpha.-glycerophosphorylcholine itself, and any possible
impurity thereof, by means of an Evaporative Light Scattering
Detector type.
10. The method according to claim 9, wherein said stationary phase
has secondary and tertiary amino groups.
11. The method according to claim 9, wherein said stationary phase
consists of particles of support material with a polymer coating
containing amino groups.
12. The method according to claim 9, wherein the eluent phases
consist of an aqueous solution, a polar organic solvent, or a
mixture thereof.
13. The method according to claim 12, wherein said aqueous solution
has a pH ranging between 3 and 6.
14. The method according to claim 12, wherein said polar organic
solvent is a C.sub.1-C.sub.4 alcohol, acetonitrile, or a mixture
thereof.
15. The method according to claim 14, wherein said C.sub.1-C.sub.4
alcohol is methanol.
Description
[0001] The object of the present invention is a process for the
purification of L-.alpha.-glycerophosphorylcholine, wherein
L-.alpha.-glycerophosphorylcholine is crystallized from DMSO or
from a mixture of DMSO with at least another solvent, preferably
selected from water, alcohol, halogenated solvents, ethers, esters
and/or amides. Such a process allows to obtain
L-.alpha.-glycerophosphorylcholine, which also constitutes an
object of the present invention, having a purity greater than
99.5%, preferably greater than 99.7%, even more preferably greater
than or equal to 99.9%; in particular, it allows to obtain GPC
contaminated by less than 0.1% by its .beta.-GPC isomer and/or by
less than 0.1% by the cyclic species cGP.
[0002] A further object of the present invention is represented by
a method for determining the purity of
L-.alpha.-glycerophosphorylcholine comprising the elution of
L-.alpha.-glycerophosphorylcholine through an HPLC column having an
amino stationary phase, and subsequent detection of
L-.alpha.-glycerophosphorylcholine itself, and any impurity
thereof, by means of an Evaporative Light Scattering Detector
type.
[0003] The current economic and demographic trends have led to a
general aging of the population, and this phenomenon is
particularly marked in the richest regions of the globe.
[0004] The decline of individual capabilities with aging has,
therefore, become a very important issue, and there is an
increasing need of identifying methods that help to slow this
decline.
[0005] In particular, the weakening of mental abilities is one of
the most negative consequences of aging, and many research efforts
are devoted to finding effective therapies to fight it.
[0006] In this area, one of the products of greatest interest is
L-.alpha.-glycerophosphorylcholine (GPC), whose structure is shown
below.
##STR00001##
[0007] GPC has a well-established use as therapeutic agent in the
field of cognitive disorders, and its worldwide consumption, based
on a wide confirmation of efficacy in medical practice and in
numerous clinical trials, is well established. The methods of
preparation of GPC can be divided into two main types: those based
on the deacetylation reaction of phosphatidylcholine of natural
origin, and those wherein GPC is synthesized from raw materials
commercially available in the market of chemical intermediates.
[0008] The methods of the first type have the advantage of using as
raw material products, such as soy lecithin, already widely used in
the food industry, and characterized in that their handling does
not generate any particular safety risks.
[0009] The procedures based on the GPC synthetic approach may
instead give rise to concerns in the management of the substances
used and, in particular, of the chiral synthetic precursors. These
are mainly R-glycidol and R-chloropropanediol (structures I and II
outlined below, respectively) and, in both cases, these are
alkylating molecules having considerable toxicity characteristics.
It is therefore clear how their use may generate issues of
dangerousness during the use and handling, and even danger in the
consumption of GPC obtained from these, in case of residual traces
or if other species were present (for example, intermediates or
by-products) maintaining their toxicity characteristics.
##STR00002##
[0010] Whatever the GPC method of preparation is, the part relating
to its purification and isolation will have a great impact on the
quality of the product, in particular its chemical purity may
influence its toxicity and therapeutic profile. Such purity is
traditionally verified through "Thin Layer Chromatography" (TLC).
This type of analysis has good versatility characteristics and
allows to evaluate the sample as a whole, since all the components,
from the most polar to the least polar, are viewable at the same
time. This analytical technique is however not entirely
satisfactory as regards the quantitative determination of any
impurities and the separation of species with similar structural
features, such as for example isomers with comparable polarity.
[0011] HPLC is the analytical technique most frequently adopted to
overcome these TLC shortcomings; in the case of GPC, the use of
HPLC resulted, however, less easy than usual due to the high
polarity characteristics of the analyte, which limit the
interaction with the stationary phases, and the absence of
chromophores which make possible the use of a UV detector, i.e. the
more prevalent detector in quality control laboratories. The
published methods are unsatisfactory for sensitivity, and for the
shape of the chromatographic signals having enlarged or asymmetric
peaks; in these conditions there is a reduction of separation
power, and there is the risk of having signals corresponding to
certain species that are hidden under other chromatographic
signals, resulting in loss of the ability to highlight any present
impurities. See, for example: J. Am. Oil Chem. Soc (2012)
89:1155-1163; Talanta (2012) 94:22-29; Journal of Chromatography A
(2012) 1220:108-114, incorporated herein by reference.
[0012] This situation is badly related with current guidelines in
the pharmaceutical field, which provide a detailed description of
the impurities profile in the production of active ingredients
intended for human use.
[0013] The development of new analytical techniques for the
determination of the quality of the different preparations of GPC
is therefore of primary importance and, in the event that the
presence of impurities in preparations of GPC was highlighted with
such analytical techniques, the definition of new standards of
purity for GPC, and new purification procedures such as to enable
the production of GPC in accordance with the current standards of
quality.
DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 HPLC-ELSD chromatogram of GPC obtained by procedure
1
[0015] FIG. 2 HPLC-ELSD chromatogram of GPC obtained from example
2
[0016] FIG. 3 HPLC-ELSD chromatogram of GPC obtained from example 1
(Comparative)
[0017] FIG. 4 .sup.31P-NMR spectrum of GPC obtained by procedure
2
[0018] FIG. 5 .sup.31P-NMR spectrum of GPC obtained from example
3
[0019] FIG. 6 HPLC-ELSD chromatogram of GPC obtained by procedure
1
[0020] FIG. 7 HPLC-ELSD chromatogram of GPC obtained from example
3
[0021] FIG. 8 HPLC-ELSD chromatogram of GPC obtained by procedure
3
[0022] FIG. 9 HPLC-ELSD chromatogram of GPC obtained from example
4
[0023] FIG. 10 HPLC-ELSD chromatogram of GPC obtained from example
5 (Comparative)
[0024] FIG. 11 HPLC-ELSD chromatogram of GPC obtained by procedure
4
[0025] FIG. 12 HPLC-ELSD chromatogram of GPC obtained from example
6 (Comparative)
DESCRIPTION OF THE INVENTION
[0026] We have now unexpectedly found that in the HPLC analysis of
GPC is possible to overcome the issues described above by suitably
employing an amino HPLC chromatographic column type, i.e. those
wherein an amino functionality is bound to the surface of the
stationary phase. Several column of this type are commercially
available: for example, Supelcosil.TM. LC-NH.sub.2, Hypersil
Gold.TM. amino, Zorbax.RTM. NH.sub.2, YMC.TM. Polyamine II,
Nucleosil.RTM. NH.sub.2, Luna.RTM. NH.sub.2. The stationary phase
may consist of particles of support material, such as for example,
silica, with a coating consisting of a polymer coating containing
amino groups, preferably secondary and tertiary amino groups. The
description of a stationary phase of this type is reported on pages
22 and 23 of the publication "HPLC Columns YMC Classics", by YMC
Europe GmbH, incorporated herein by reference.
[0027] At the same time, an Evaporative Light Scattering Detector
(ELSD) type will have to be used.
[0028] Such detectors are described, for example, in Anal. Chem
(1997) 69:561A-563A and U.S. Pat. No. 6,229,605, incorporated
herein by reference. These are tools in which the eluate is sent
into an evaporation chamber where the solvent is evaporated to
leave a mist of tiny sample particles that scatter a light beam.
The detector response is proportional to the diffusion which is
dependent on the amount of sample present; this system makes
possible the detection of species devoid of chromophores, such as
GPC, also while using not isocratic elution methods.
[0029] With this combination of stationary phase and detector,
various eluents may be used and, in particular, systems suitably
consisting of aqueous buffer systems, such as, for example,
ammonium acetate buffer, possibly in combination with organic
solvents such as, for example, methanol and/or acetonitrile. Such
eluent systems may be used in isocratic mode, i.e. by keeping the
same eluent for the entire duration of the chromatographic run, or
in gradient mode, wherein the composition of the eluent varies
during the chromatography according to a predetermined program. The
optimization of the method of analysis will be performed following
the teachings of the art.
[0030] An object of the present invention is therefore a method for
determining the purity of L-.alpha.-glycerophosphorylcholine
comprising the elution of L-.alpha.-glycerophosphorylcholine
through an HPLC column having an amino stationary phase, and
subsequent detection of L-.alpha.-glycerophosphorylcholine itself,
and any impurity thereof, by means of an Evaporative Light
Scattering Detector type.
[0031] The eluent phase or phases may consist of an aqueous
solution, a polar organic solvent, or a mixture thereof; the
aqueous solution preferably has a pH ranging between 3 and 6, even
more preferably between 4 and 5. The polar organic solvent may be a
C.sub.1-C.sub.4 alcohol, acetonitrile, or a mixture thereof; the
C.sub.1-C.sub.4 alcohol is preferably methanol.
[0032] This combination allows to overcome the defect of the
enlargement of the chromatographic peak corresponding to GPC, and
at the same time allows to carry out the determination of GPC
concentration with high sensitivity.
[0033] At the same time, it was found that it is possible to detect
the presence in GPC preparations of other species deriving from the
process of preparation of GPC starting from soya lecithin.
[0034] Among these species, there are included
L-.alpha.-glycerophosphoethanolamine (GPE), sugars, and analogues
thereof, deriving from their presence, or the presence of one of
their precursors, in the soya lecithin used as raw material in the
GPC production process.
[0035] Having obtained this important result, it was of immediate
interest to verify in a more sensitive and selective way than in
the past, the impurities profile, if present, in samples of GPC
obtained by production methods most suitable for the pharmaceutical
product for human use.
[0036] For this purpose, semi-synthetic processes based on soya
lecithin deacylation were mainly considered, being the fully
synthetic procedure judged to be unsuitable for the production of
GPC for human use, in view of the potential contamination with
highly toxic raw materials, such as glycidol or chloropropanediol,
that are used in these procedures. While taking into consideration
said contraindications related to the production of GPC by a fully
synthetic route, a GPC sample of Chinese origin, produced under
this approach, was submitted to the new analytical procedure, for a
complete evaluation.
[0037] The samples deriving from semi-synthetic procedures were
generated following the procedures reported in WO9013552
(specifically the Example 2) and EP575717 (specifically the Example
1). In these documents, procedures based on the purification of GPC
with ion exchange resins are reported, and they do not involve the
precipitation of GPC as adduct with cadmium chloride
(GPC.CdCl.sub.2) and silica chromatography; these are therefore
methods that, avoiding the use of toxic cadmium salts and procedure
characterized by low productivity, are really applicable to GPC
large scale production.
[0038] The analysis results showed, in all the samples, the
presence of a peak eluting before and with a retention time very
close to that of GPC, having a RRT (Relative Retention Time) equal
to about 0.94. This signal, of significant intensity, does not
correspond to species that it would have been reasonable to expect
in view of the preparation process, such as glycerol, GPE,
glycerophosphoric acid, or sucrose.
[0039] Given that the chromatographic behavior of the new detected
species was so similar to that of GPC, as not to have been
highlighted until now, and that even with the new HPLC method
object of the present invention it differed very little from that
of GPC, we then hypothesized that it corresponded to a positional
isomer thereof.
[0040] The chemical structure of the beta isomer of GPC
(.beta.-GPC) is reported below.
##STR00003##
[0041] .beta.-GPC is not commercially available and, in order to
check whether .beta.-GPC could be the species detectable with the
new method, its synthesis was carried out according to the scheme
illustrated below.
##STR00004##
[0042] .beta.-GPC thus obtained was analyzed by NMR, to confirm the
structure, and by HPLC showing that not only its purity was high,
but also that its retention time was compatible with that of the
unknown species revealed by analyzing the different GPC samples. It
was therefore possible to attribute to the unknown signal a
correspondence to the .beta.-GPC structure, and this attribution
was confirmed by HPLC "spiking" experiments, wherein to a GPC
sample showing the presence of the species with a RRT at about 0.94
in HPLC, calibrated amounts of .beta.-GPC were added, observing the
corresponding increase of the signal with RRT at about 0.94 without
a splitting thereof.
[0043] As further confirmation of the structure attribution,
.sup.31P-NMR experiments were performed that allowed the
identification of a signal at about -0.6 ppm in the samples wherein
the presence of the species with RRT at about 0.94 was higher. The
signal of the .beta.-GPC synthesized following the scheme reported
above was revealed at the same field value.
[0044] The .sup.31P-NMR method is therefore a possible alternative
to the HPLC procedure, however, due to its lower sensitivity and
the wider availability of HPLC instrumentation in quality control
laboratories, the HPLC method object of the present invention
remains preferable for the determination of the species having RRT
of about 0.94 (.beta.-GPC).
[0045] By means of .sup.31P-NMR analysis it was also possible to
identify in the spectrum of the product obtained according to
EP575717 an additional signal at about 18.4 ppm. These values are
compatible with those of the cyclic phosphate cGP, illustrated
below in the form of the sodium salt.
##STR00005##
cGP (Sodium Salt)
[0046] The formation of this cyclic phosphate is conceivable in the
conditions of isolation of GPC according to the procedure of
EP575717. To support this hypothesis, an authentic sample of cGP
was prepared according to the scheme illustrated below.
##STR00006##
[0047] .sup.31P-NMR analysis of this product confirmed the
assignment, showing the same signal at about 18.4 ppm and the
absence of splitting of the signal in appropriate spiking
experiments.
[0048] In the light of what was found with the innovative
analytical methods that represent one embodiment of the invention,
we have experimentally verified whether the known GPC purification
methods could adequately remove the highlighted impurities.
[0049] Excluding, for the reasons already expressed, the
purification by complexation with cadmium chloride and
chromatography on silica, we have identified the crystallization
from ethanol as the best procedure, in terms of industrial
applicability, among those reported in the prior art (see for
example WO9013552). The sample obtained according to the procedure
of WO9013552 was crystallized from ethanol, but the HPLC analysis
showed that the purification was only partial, being a significant
amount of beta isomer (0.48%) still present in the product.
[0050] The following Table 1 summarizes the analytical results of
the GPC samples obtained following the different preparation
procedures.
TABLE-US-00001 TABLE 1 .beta.-GPC cGP Preparation (area % by (area
% by .sup.31P- Sample procedure HPLC) NMR) Note Procedure 1
WO9013552 0.8% Procedure 2 EP575717 0.1% 0.2% Example 1 0.48%
Crystallization from ethanol of (Comparative) the sample from
procedure 1 GPC Synthesis 1.8% Commercial GPC produced in
commercial China. Several other impurities present in a total
amount of 3% as area % by HPLC
[0051] A new system for the purification of GPC has now been
identified, and this finding also represents an embodiment of the
invention.
[0052] The new purification is based on the use of dimethyl
sulfoxide (DMSO). For the realization of the invention, this
solvent may be used in different conditions and in different
quantitative ratios with respect to the GPC to be purified.
[0053] In the embodiment of the invention, in addition to DMSO,
other solvents may be present, such as polar, of medium polarity or
non-polar solvents. These additional solvents may belong to
different classes of solvents such as, but not limited to,
halogenated solvents, alcohols, ethers, esters and amides.
[0054] Examples of solvents that can be employed for the
realization of the invention are, but not limited to, water,
methanol, ethanol, isopropanol, n-butanol, methylene chloride,
tetrahydrofuran, ethyl acetate, and DMF.
[0055] According to an aspect of the invention, DMSO is used in an
amount ranging between 2 and 100 parts by volume per one part by
weight of L-.alpha.-glycerophosphorylcholine, preferably between 3
and 70, more preferably ratios ranging between 4 and 30, more
preferably ratios ranging between 5 and 15 parts by volume.
[0056] According to a further aspect of the invention, any solvent
or any additional solvents are used in a quantity ranging between
0.01 and 10 volumes per volume of DMSO, preferably from 0.05 to 5,
more preferably ratios ranging between 0.1 and 1.
[0057] Depending on the process parameters, a proper
crystallization of GPC may be obtained, or the purification of GPC
crystals containing impurities may also be performed by suspending
them in DMSO, using suitable combinations of time and temperature,
and then separating the crystals from the liquid phase; also in
this case other solvents may be present in combination with
DMSO.
[0058] The purification may be performed at different temperatures;
as usual, it may be convenient to adopt different temperatures in
the different stages of the purification process, having a lower
temperature in the phase of final isolation of GPC.
[0059] Suitable temperatures for the conduction of the purification
may be between 100.degree. C. and 0.degree. C., preferably between
70.degree. C. and 5.degree. C., more preferably between 50.degree.
C. and 15.degree. C.
[0060] In choosing the temperature, it would be preferable to take
into account the freezing temperature of DMSO, which can vary
mainly depending on the presence of additional solvents, and the
concentration of GPC and other species present. For the isolation
of GPC, equipment known to the person skilled in the art may be
used, such as centrifuges or filtration systems, closed or open.
The crystals may be directly subjected to a washing procedure using
the same solvent, or the same mixture of solvents, used for the
purification; alternatively, for washing the product, solvents or
solvent mixtures other than those used in the crystallization or
suspension step may be used.
[0061] The ratio of DMSO to be used compared to the quantity of GPC
used in the purification process object of the invention may vary
considerably, and may conveniently be chosen according to the
teachings of the art. In practice, in order not to reduce more than
required the process productivity, it will be convenient to use not
too high quantities of DMSO and, on the other hand, these
quantities should not be excessively reduced to avoid creating
difficulties in the operations of separation of the crystallized
product. DMSO and GPC ratios ranging between 2 and 100 parts by
volume (liters) per parts by weight (kg) may be considered
convenient for this embodiment, more preferably ratios ranging
between 3 and 70, more preferably ratios ranging between 4 and 30,
more preferably ratios ranging between 5 and 15 parts by volume per
part by weight of GPC.
[0062] A particularly advantageous mode to carry out this invention
is the crystallization of GPC from its solutions in water, or other
solvents, or solvent mixtures. By adding DMSO to such solutions, it
was found that solutions that are stable for a time sufficient to
conduct the distillation operations without concurrent
precipitation of GPC are obtained. Exploiting the higher boiling
temperature of the DMSO with respect to the other solvents commonly
used, it is therefore possible to modify, without obstacles caused
by the simultaneous precipitation of the product, the composition
of the mixture of solvents used to perform the crystallization.
[0063] Once the solvents mixture composition has been achieved, it
is possible to induce the crystallization of GPC by addition of an
aliquot of crystallized GPC, that will act as a trigger. This
option does not appear to be required; when its application is
desired, a sample of GPC crystals may be used as a trigger which
should not necessarily have a particularly high purity degree,
since a close dependence between the quality of the trigger used
and that of the GPC crystals isolated at the end of the
purification was not observed.
[0064] It was surprising to observe how these purification
procedures using DMSO allow to obtain high purity GPC, even from
preparations containing significant amounts of other molecular
species, including those previously described.
[0065] In particular, the ability of the purification system object
of the present invention to remove the GPC beta isomer appears
significant and unpredictable, since it has chemical-physical
characteristics very similar to those of the product whose
isolation with a high degree of purity is desired.
[0066] Unlike the crystallization from ethanol, it was also found
that the purification method object of the invention allows to
purify GPC from some of the species most frequently found in GPC
preparations obtained from lecithins.
[0067] In particular, it was found in embodiments of the invention
that it is possible to effectively remove GPC impurities such as
sucrose and GPE.
[0068] Table 2 below summarizes the main results achieved in these
experiments in comparison with those relating to comparative
experiments performed using ethanol as the crystallization
solvent.
TABLE-US-00002 TABLE 2 Impurities Residual content present in GPC
of impurity in used as test sample crystallized Content GPC
Crystallization (area % by (area % by Example solvent Impurity
HPLC) HPLC) Note 1 Ethanol .beta.-GPC 0.8% 0.48% GPC
crystallization (Comparative) obtained by procedure 1 2 DMSO
.beta.-GPC 0.8% 0.16% GPC crystallization obtained by procedure 1 3
DMSO .beta.-GPC 0.1% <0.01% GPC crystallization cGP 0.2%*
<0.01%* obtained by procedure 2 4 DMSO GPE 0.2% <0.01% GPC
crystallization obtained by procedure 3 5 Ethanol GPE 0.2% 0.02%
GPC crystallization (comparative) obtained by procedure 3. Low
yield. 6 DMSO Sucrose 2% <0.01% GPC crystallization obtained by
procedure 4. 7 Ethanol Sucrose 2% 0.14% GPC crystallization
(comparative) obtained by procedure 4. Low yield *.sup.31P-NMR
measurement
[0069] The results shown in Table 2 highlight the usefulness of the
embodiments of the invention in the purification of GPC. In this
regard, it is noteworthy the fact that, as in Example 6, the
realization of the invention demonstrates the possibility to
crystallize GPC also in the presence of sugars, in this case
sucrose, and the high purification efficiency achievable even in
these cases. Conversely, the GPC crystallization from ethanol did
not allow satisfactory removal of sucrose. Moreover, GPC, and in
even greater measure sugars such as sucrose, behave as inhibitors
of crystallization when ethanol is used as the crystallization
solvent; consequently, in these cases the crystallization yields
are so low as to render the procedure impractical in processes for
the industrial production of GPC.
[0070] A further embodiment of the invention consists in GPC whose
purity is greater than 99.5%, preferably greater than 99.7%, even
more preferably with a purity greater than or equal to 99.9%.
[0071] A specific feature is represented by a GPC contaminated by
less than 0.1% by its .beta.-GPC isomer, and a GPC contaminated by
less than 0.1% by the cyclic species cGP.
[0072] For the purposes of the present invention, the purities
indicated above are measured as the percentage areas with HPLC
methods and/or .sup.31P-NMR and, preferably, with HPLC methods
and/or .sup.31P-NMR described and exemplified in Examples 8 and 9,
respectively.
[0073] The following examples are intended to illustrate some of
the methods of the invention without in any way limiting it.
EXAMPLES
Procedure 1
Preparation of GPC According to Example 2 of WO9013552
[0074] To 125 g of soya lecithin, 500 mL of methanol are added and
kept under stirring until complete dissolution. 20 mL of 30% sodium
methylate in methanol are added. Stirring is continued at room
temperature for 3 hours. After filtration, the residue is washed
with methanol (3.times.10 mL). The filtrate is neutralized (pH of
about 6) with glacial acetic acid, then it is concentrated to a
residual volume of about 125 mL, the upper oily phase is separated.
The enriched lower phase is eluted on a column containing 140 mL of
Amberlyst 15 resin (in acid form) set in methanol. The elution is
progressed with 375 mL of methanol, followed by 300 mL of
water.
[0075] The aqueous eluate is eluted on a sequence of three
chromatographic columns thus prepared: the first with 30 mL of IR
93 resin in OH-- form set in water, the second with 30 mL of IR 401
resin in OH-- form set in water, the third with 12 mL of IRC 50
resin in acid form set in water.
[0076] The final eluate is concentrated to a residue viscous fluid
with a water content equal to 15%.
[0077] The HPLC-ELSD analysis showed a content of .beta.-GPC equal
to 0.8% (area percent).
Procedure 2
Preparation of GPC According to Example 1 of EP575717
[0078] 200 g of the fraction enriched in phosphatidylcholine
obtained commercially by extraction of soy lecithin are dissolved
in 600 mL of methanol. The obtained solution is eluted on a
chromatographic column containing Duolite A 147 resin in basic
form, set in methanol. After loading of the enriched solution, GPC
elution is completed with methanol. The eluate is neutralized with
acetic acid, and concentrated to a residual volume of 300 mL
obtaining a sharp stratification of two phases. The enriched lower
phase is separated, diluted with methanol, and extracted twice with
200 mL of n-heptane.
[0079] 800 mL of n-butanol are added to the enriched lower phase,
and the solution is concentrated to a residual volume of about 300
mL, cooled to T.about.5.degree. C., and filtrated. The GPC crystals
are dissolved in 30 mL of demi water, and the solution is
concentrated up to a viscous liquid with a residual content of
water equal to 15%.
[0080] The HPLC-ELSD analysis showed a .beta.-GPC content equal to
0.1% (area percent).
[0081] The .sup.31P-NMR analysis showed a cGP content equal to 0.2%
(area percent).
Procedure 3
Preparation of GPC Contaminated with GPE
[0082] GPE in crystals is added to a solution of GPC in water, the
solution is concentrated up to a viscous liquid with a residual
content of water equal to 15%.
[0083] The HPLC-ELSD analysis showed a GPE content equal to 0.2%
(as area percent).
Procedure 4
Preparation of GPC Contaminated with Sucrose
[0084] Sucrose in crystals is added to a solution of GPC in water,
the solution is concentrated up to a viscous liquid with a residual
content of water equal to 15%.
[0085] The HPLC-ELSD analysis showed a sucrose content equal to 2%
(as area percent).
Example 1 (Comparative)
Purification of GPC Obtained by Procedure 1 Using Crystallization
from Ethanol
[0086] 2.5 g of GPC in the form of a viscous fluid (obtained by
procedure 1) are dissolved in 5 mL of ethanol under stirring at
T.about.50.degree. C. The solution is cooled down to
T.about.10.degree. C. and triggered with a few GPC crystals, in a
few minutes the formation of a precipitate is observed. It is
cooled down to T.about.5.degree. C. and kept under stirring between
0.degree. C. and 5.degree. C. for 1.5 hours, the crystals are
filtered through a Buchner funnel, washing with ethanol. After
drying under vacuum, 1.4 g of GPC are obtained.
[0087] The HPLC-ELSD analysis showed a .beta.-GPC content equal to
0.48% (as area percent).
Example 2
Purification of GPC Obtained by Procedure 1 Using Crystallization
from DMSO
[0088] To 2.5 g of GPC in the form of a viscous fluid (obtained by
procedure 1), 14 mL of DMSO are added, the mixture is warmed up to
T.about.50.degree. C. under stirring, it is cooled down to
T.about.25.degree. C. and triggered with a few GPC crystals. It is
kept under stirring between 20.degree. C. and 25.degree. C. for 16
hours; during this time the formation of a precipitate is observed,
the crystals are filtered through a Buchner funnel, washing with
ethanol. After drying under vacuum, 1.9 g of GPC are obtained.
[0089] The HPLC-ELSD analysis showed a .beta.-GPC content equal to
0.16% (area percent).
Example 3
Purification of GPC Obtained by Procedure 2 Using Crystallization
from DMSO
[0090] To 11.2 g of GPC in the form of a viscous fluid (obtained by
procedure 2), 60 mL of DMSO are added, the mixture is warmed up to
T.about.50.degree. C. under stirring, it is cooled down to
T.about.25.degree. C. and triggered with a few GPC crystals. It is
kept under stirring between 20.degree. C. and 25.degree. C. for 16
hours; during this time the formation of a precipitate is observed,
the crystals are filtered through a Buchner funnel, washing with
ethanol. After drying under vacuum, 8.2 g of GPC are obtained.
[0091] The HPLC-ELSD analysis showed the absence of .beta.-GPC
[0092] The .sup.31P-NMR analysis showed the absence of cGP
Example 4
Purification of GPC (Contaminated with GPE) Using Crystallization
from DMSO
[0093] To 10.7 g of GPC in the form of a viscous fluid (obtained by
procedure 3) 20 mL of DMSO are added, the mixture is warmed up to
T.about.70.degree. C. under stirring, 80 mL of DMSO are added, it
is cooled down to T.about.25.degree. C. and triggered with a few
GPC crystals. It is kept under stirring between 20.degree. C. and
25.degree. C. for 16 hours; during this time the formation of a
precipitate is observed, the crystals are filtered through a
Buchner funnel, washing with ethanol. After drying under vacuum,
8.7 g of GPC are obtained.
[0094] The HPLC-ELSD analysis showed the absence of GPE
Example 5 (Comparative)
Purification of GPC (Contaminated with GPE) Using Crystallization
from Ethanol
[0095] 11.2 g of GPC in the form of a viscous fluid (obtained by
procedure 3) are dissolved in 20 mL of ethanol under stirring at
T.about.45.degree. C. The solution is cooled down to
T.about.5.degree. C. and triggered with a few GPC crystals. It is
kept under stirring between 0.degree. C. and 5.degree. C. for 3
hours; during this time the formation of a precipitate is observed,
the crystals are filtered through a Buchner funnel, washing with
ethanol. After drying under vacuum, 3.4 g of GPC are obtained.
[0096] The HPLC-ELSD analysis showed a GPE content equal to 0.02%
(as area percent).
Example 6
Purification of GPC (Contaminated with Sucrose) Using
Crystallization from DMSO
[0097] To 11 g of GPC in the form of a viscous fluid (obtained by
procedure 4) 20 mL of DMSO are added, the mixture is warmed up to
T.about.70.degree. C. under stirring, 80 mL of DMSO are added, it
is cooled down to T.about.25.degree. C.; during this time the
formation of a precipitate is observed. It is kept under stirring
between 20.degree. C. and 25.degree. C. for 16 hours, the crystals
are filtered through a Buchner funnel, washing with ethanol. After
drying under vacuum, 7.8 g of GPC are obtained.
[0098] The HPLC-ELSD analysis showed the absence of sucrose
Example 7 (Comparative)
Purification of GPC (Contaminated with Sucrose) Using
Crystallization from Ethanol
[0099] 11.0 g of GPC in the form of a viscous fluid (obtained by
procedure 4) are dissolved in 20 mL of ethanol under stirring at
T.about.45.degree. C. The solution is cooled down to
T.about.5.degree. C. and triggered with a few GPC crystals. It is
cooled down to T.about.5.degree. C. and kept under stirring between
0.degree. C. and 5.degree. C. for 2.5 hours; during this time the
formation of a precipitate is observed. The crystals are filtered
through a Buchner funnel, washing with ethanol. After drying under
vacuum, 1.1 g of GPC are obtained.
[0100] The HPLC-ELSD analysis showed a sucrose content equal to
0.14% (as area percent).
Example 8
[0101] GPC analysis method by HPLC-ELSD. Operating parameters: HPLC
column: YMC.TM. Polyamine II 250.times.4.6 mm (5 .mu.m) Oven
temperature: 35.degree. C. Flow: 0.7 mL/min Eluent phases: Phase A:
85% (acetonitrile 75%, methanol 25%)
[0102] 15% (50 mM ammonium acetate buffer pH 4.5)
Phase B: 65% (acetonitrile 75%, methanol 25%) 35% (50 mM ammonium
acetate buffer pH 4.5) Gradient program:
TABLE-US-00003 minutes Phase A % Phase B % 0 98 2 5 98 2 27 2 98 32
2 98 33 98 2 45 98 2
Detector ELSD
[0103] Detector parameters: Nitrogen flow=1.4 mL/min;
Temperature=90.degree. C.; Gain=1 Samples preparation: dilution of
GPC samples to about 20 mg/mL in methanol. Injection volume: 20-40
.mu.L NOTE: The baseline fluctuations that are observed up to
RT.about.5 minutes are present in the blank (not due to substances
present in the sample).
[0104] In the following table the reference values for the elution
of the species of interest are shown.
TABLE-US-00004 RRT Chemical RT Relative retention time with species
Retention Time respect to GPC GPC ~16.2 min 1 .beta.-GPC ~15.3 min
~0.94 GPE ~31.5 min ~1.94 sucrose ~22.0 min ~1.36
[0105] The limit of detection (LOD) of the impurities analyzed
using this method is less than 0.01% with respect to GPC
Example 9
GPC Analysis Method by .sup.31P-NMR
[0106] Instrument NMR Varian VXR-200 or instruments at least
equivalent Sample preparation 0.8 g of GPC to be analyzed are
dissolved in 0.8 mL of D.sub.2O and the solution is charged into
the NMR tube Temperature Ambient temperature Number of acquisitions
Not less than 400
[0107] In the following table the reference values for the chemical
shift of the species of interest are shown.
TABLE-US-00005 .sup.31P-NMR chemical shift Chemical species (ppm)
GPC -0.1 .beta.-GPC -0.6 GPE 0.4 cGP 18.4
[0108] The limit of detection (LOD) of the impurities analyzed
using this method is less than 0.01% with respect to GPC
* * * * *