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Journal of Liquid
Chromatography & Related
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http://www.tandfonline.com/loi/ljlc20
Development of An HPLC-UV
Method for Determination of
Taurine in Infant Formulae and
Breast Milk
I. M. P. L. V. O. Ferreira
a
, M. V. Nunes
a
, E. Mendes
a
, F. Remião
b
& M. A. Ferreira
a
a
CEQUP
b
Laboratório de Bromatologia Laboratório de
Toxicologia Faculdade de Farmácia, Universidade do
Porto Rua Aníbal Cunha , 164 4050, Porto, Portugal
Published online: 23 Sep 2006.
To cite this article: I. M. P. L. V. O. Ferreira , M. V. Nunes , E. Mendes , F. Remião
& M. A. Ferreira (1997) Development of An HPLC-UV Method for Determination of
Taurine in Infant Formulae and Breast Milk, Journal of Liquid Chromatography &
Related Technologies, 20:8, 1269-1278, DOI: 10.1080/10826079708010975
To link to this article: http://dx.doi.org/10.1080/10826079708010975
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Downloadedby[UniversityofWestFlorida]at06:1103October2014
J. LIQ. CHROM. & REL. TECHNOL., 20(8), 1269-1278 (1997)
DEVELOPMENT OF AN HPLC-UV METHOD
FOR DETERMINATION OF TAURINE IN INFANT
FORMULAE AND BREAST MILK
I. M. P.L. V. 0.Ferreira,t M. V. Nunes, E. Mendes,
F. Remiilo,I M. A. Ferreirat
~CEQUP
tLaboratoriode Bromatologia
Laboratbriode Toxicologia
Faculdade de Farmacia, Universidade do Porto
Rua Anibal Cunha 164
4050 Porto, Portugal
ABSTRACT
A rapid and accurate hgh performance liquid chromato-
graphic (HPLC)procedurewas proposed for routine and selective
determination of taurine in infant formulas and breast milk. The
sample preparation was simple, involving protein removal and
filtration operations. Afterwards, taurine was derivatised with 0-
phthalaldehyde/2-mercaptoethanol prior to injection onto a
reversed phase column CI8 (SloODS2). Isocratic elution was
carried out using 0.05 M phosphate buffer pH 5.3/ methanol
(60:40) mixture. The effluentwas monitored by a UV detector at
350 nm. A linear relationship was found between peak area and
a concentration range of 1-70 pg/mL. The detection limit was
0.3 pg/mL. Effective separation and quantification was achieved
in under six minutes. No interference of other amino acids was
observed.
1269
Copyright 8 1997by Marcel Dekker. Inc.
Downloadedby[UniversityofWestFlorida]at06:1103October2014
1270 FERREIRA ET AL.
Extensive validation of the proposed method was carried out
both by the standard additions method and by comparison with a
fluorimetric method of known accuracy. The precision was better
than 1.4%.
INTRODUCTION
Taurine. 2-aminoethanesulphonic acid. is a sulphur-containing p-amino
acid that is not incorporated into proteins. However, it takes part in
biochemical reactions of major importance, such as conjugation with bile acids
to form bile salts essential for fat absorption, cell membrane stabilization,
antioxidation. detoxification. osmoregulation, neuromodulation, and brain and
retinal development.’
Considerable evidence has accumulated that neonates and infants, who
have a very limited ability to synthesizetaurine, are especially prone to develop
taurine deficiency and that they depend on an external taurine supply.’
Taurine concentrations in milk are very variable, depending on species.
Wlule breast milk is an excellent source of taurine in the developing infant.
cow’s milk has a very low taurine content. Nowadays the supplementation of
infant formulae with taurine in concentrations similar to those found in human
milk is therefore re~ommended.~.‘
Many methods for measuring taurine in several matrices viz. biological
samples, such as tissue homogenates, urine, and serum have been published,
including gas chromatography’ and HPLC analysis of diferent amino acid
derivatives.6-” One of the most sensitive and commonly used derivatising
agent techniques is the use of o-phthaldehyde / mercaptoethanol, which,
originates a fluorescent adduct that enables the fluorimetric determination of
taurine.6*1’
Other HPLC fluorimetric determinations in similar matrices include
derivatising reaction with hamine*and fluore~camine.~
It is also possible to use UV absorbance detection at 350 nm with different
derivatisation techniques. includmg o-phthaldehyde / mercaptoethanol’ and
dinitrofluorobenzene.‘~,’~Interestingly. research work developed by Chen at
aI.l2is distinguished from the rest since one of the matrices used was human
milk.
Downloadedby[UniversityofWestFlorida]at06:1103October2014
TAURINE IN W A N T FORMSJLAE AND BREAST MILK 1271
Some of these methods require time-consuming pre-treatment of the
samples. in order to eliminate interfering substances or complicated
derivatising treatment of the samplebefore assay.
This paper describesa rapid and accurate method for the determination of
taurine in infant formulae and breast milk. Although, the sample matrix is of
complex nature, the pre-treatment applied is simple and the method is suitable
for rapid routine assays of a large number of samples and uses a UV detector
which is much more common in control laboratories than the fluorimetric
detector.
MATERIALS
Apparatus
The chromatographic analysis was carried out in a Gilson, high
performance liquid Chromatograph (Gilson Medical Electronics, Villiers le
Bel, France) equipped with a type305 pump, a type 302 pump and a type 7125
Rheodyne Injector with a 20 p1 loop. A Gilson 118, variable long wave ultra
violet detector was also used.
The chromatographic separation was achieved with a Spherisorb Slo
ODs2Chromatographiccolumn, 10 pm. The integrator used was a Gilson 712
HPLC System Controller Software.
A Gilson 121 Spectrofluorometerwas used for detection of the fluorescent
adduct.
Reagents and Standards
Taurine and o-phthaldehydewere obtained from Sigma Chemical Co. L-
Amino acids kit was also from Sigma (Kit No. LAA-21). Sulphosalicylicacid,
boric acid, sodium hydroxide and mercaptoethanol,all p.a., were obtained from
Merck. Methanol (LiCrosolv)was Merck "gradient grade".
Water used for chromatography possessed a resistance greater than 15
Ma. and was filtered through a membrane of 0.45 pm porosity which was
subsequentlydegassed.
Downloadedby[UniversityofWestFlorida]at06:1103October2014
1272 FERREIRA ET AL.
Sample Preparation
After homogenisation. 5.0 g of infant formulae were dissolved in 40 mL
of warm (40°C) water. 5.0 mL of the infant formulae solution or breast milk
were added to 5.0 mL of sulphosalicylic acid solution 0.2 M. mixed thorougly.
and allowed to stand for 10 min. All samples were filtered through W42 paper
and thereafter. through 0.2 pm filter paper.
The use of sulphosalicylic acid as a protein precipitating agent has been
shown to result in a higher YOrecovery of taurine.”
Denvatisation Procedure
Taurine was derivatised with o-phthalaldehyde (OPA, 40 mg, 0.8 mL
absolute ethanol) and 2-mercaptcethanol (4Opl) in 0.5 M borate buffer (10 mL,
3.1 g boric acid in 90 mL water. adjusted to pH 10.4with 5M NaOH. and made
up to 100 mL)6prior to injection onto a reversed phase column CIS(SloODS2).
The derivatizing solution was prepared daily and filtered through a 0.20
pm filter before use.
A 100 pL volume of standard or sample solution (after preparation) was
placed in an eppendorf and 100 pl of derivatizing reagent were added. The
derivatization reaction was allowed to proceed for exactly 1.5 min, at which
time an aliquot was injected into the HPLC.
Chromatography
The HPLC elution required a mixture of two solvents. Solvent A. 0.05 M
phosphate buffer pH 5.3. and solvent B. methanol. The two solvents were
filtered and degassedbefore use. Isocratic elution was carried out at a flow rate
of 1.5 mL/min, using 60% solvent A combined with 40 YOsolvent B. The
injection volume was 20 p1 and the chromatographic analysis was conducted at
ambient temperature.
Taurine concentration was determined at an absorbance of 350 nm. which
is the maximum wavelength on the excitation spectrum of OPA-derivatized
amino acids. Taurine peak was identified by coelution with a standard and by
comparison of the retention time.
Downloadedby[UniversityofWestFlorida]at06:1103October2014
TAURINE IN INFANT FORMULAE AND BREASTMILK 1273
Figure 1. Standard curves for taurine. Taurine was derivatised and chromatographed
by the method described in the text. Each point represents the average of triplicate
determinations. (a) Sensitivityof 0.06 AUFS, detection limite 1 p g / d . (b) Sensitivity
of 0.03AUFS, detection liniite 0.3 pg/mL.
RESULTS
Analytical Curve and Detection Limit
Under the assay conditions described, a linear relationship between the
concentrationof taurine and the UV absorbance at 350 nm was obtained. This
linearity was mantained over the concentration range 3-70 pg/mL with a
sensitivityof 0.06AUFS and 1-10 pg/mL with a sensitivity of 0.03 AUFS. The
detection limit value was calculated as the concentrationcorrespondingto three
times the standard deviation of the background noise and was 1 pg/mL and 0.3
pg/mL, respectively. The calibrationcurvesfor taurine are ilustratedin Fig. 1.
Downloadedby[UniversityofWestFlorida]at06:1103October2014
1274 FERREIRAET AL.
....
Figure 2. Typical chromatogram obtained for taurine extracted from infant formulae.
Retention time of taurine 4.51.
Triplicate determinations were made on each of the calibration standards,
the peak area values (arbitrary units) that were plotted are average values. The
relative percent average deviationsof triplicates were less than 3 YOin all cases.
Validity of the Method
The repeatability of the derivatization procedure was examined. A
standard taurine solution was derivatized and chromatographed six times.
When reaction and chromatographic conditions were carefidly optimized, the
standard deviationwas 0.02 (n=6 and concentrationof taurine 20 pg/mL).
Downloadedby[UniversityofWestFlorida]at06:1103October2014
T A W IN INFANTFORMULAE AND BREASTMILK 1275
Table 1
Recovery of Taurine from Spiked Infant Formulae
&mL pg/mL Standard CV% Recovery
Added Found’ Deviation YO
2.00 2.03 M.05 2.46 101.5
Sample 1 4.00 3.92 M.08 2.04 98.0
6.00 5.82 M.09 1.54 97.0
2.00 1.99 34.03 1.51 99.5
Sample9 4.00 4.10 k0.07 1.71 102.5
6.00 5.73 34.06 1.05 95.5
“Meanvalue found for 3 assays for each studiedconcentrations.
The interference of other amino acids was examined nest. For this
purpose, different amino acids were injected after derivatization with OPA.
They included L-Alanine, L-Valine, L-Leucine, L-Isoleucine. L-Proline. L-
Phenylalanine,L- Tryptophan, L- Methionine, Glycine, L-Serine, L-Threonine,
L-Tyrosine, L-Asparagine. L-Glutamine. L-Aspartic acid. L-Glutamic acid. L-
Lysine, L-Arginine, L-Histidine. Different retention times were obtained for
each amino acid.
Fig. 2 shows a typical chromatograph of taurine extracted from infant
formulae. Peaks corresponding to several amino acids were detected in the
chromatograms obtained for infant formulae determinations, but as shown,
these peaks were well separated from those of taurine under the conditions
used. A similar behaviourwas observedwith regards to breast milk.
The precision of the analytical method was evaluated by measuring the
peak chromatographicarea 10times on the same sample. The relative standard
deviation (RSD) ranged between 0.7 and 1.4 YO(concentration of taurine in
infant formula 42.6 and 28.6 mg of taurineA00 g of infant formula.
respectively).
Recovery studies were carried out on two different samples of infant
formulae, sample 1 with high concentration of taurine, and sample 9 with very
low concentration of taurine. The samples were analysed in triplicate before
and after the addition of known amounts of taurine and they were spiked with
Downloadedby[UniversityofWestFlorida]at06:1103October2014
1276 FERREIRA ET AL.
Table 2
Comparison Between Taurine Determined in Infant Formulae and Breast
Milk by HPLC-UV and a Comparison Method of Known Accuracy'
Taurine Determined (mg/100 g of Infant Formula)
Sample
No. HpLC-UV(y) Comparison method (x)
1
2
3
4
5
6
7
8
9
10
39.4 f0.7b
34.4 f 1.3b
28.6 f0.3b
39.0 f 0.9b
38.0 f 0.2b
44.7 f0.7b
32.7 *0.9b
30.2 f l.lb
1.O1 f 0.08"
3.99 f O.2lC
40.6 f 0.5
32.7 f0.2
27.7 *0.7
10.3k 0.5
38.8 f 0.6
43.1 +0.3
32.9 f0.1
31.0 f 0.9
0.80 ? 0.04
4.32 k0.11
Taurine Determined (mg/100 mL of Breast Milk)
11 4.13 f0.06b
12 4.25 kO.Ogb
13 2.28 f0.1lb
14 2.18 f0.09b
4.23 f 0.10
4.32 f0.05
2.43 f0.08
2.08 f0.12
aValuesare expressed as mean fstandard deviation of three determinations.
Sensitivity of 0.06 AUFS.
"Sensitivityof 0.03 AUFS.
Samples 1 to 10are from infant formulae, samples 11 to 14 are from breast
milk.
b
three different concentrations of taurine. The results are listed in Table 1. The
addition of taurine to the infant formulae was made before the protein removal.
Thus, this procedure proved the effectiveness of the extraction step and the
accuracy of the proposed method. Recovery values were between 95.5 and
102.5%. which confirm no interference effects due to matrix composition.
Downloadedby[UniversityofWestFlorida]at06:1103October2014
TAURINJ?IN INFANT FORMULAE AND BREAST MILK 1277
In order to validate the accuracy of the analytical method and because
there was no certified reference material available, not only the standard
addition method was made. but also a comparison with a method of known
accuracy." The results of these determinations of taurine in infant formulae
and breast milk are presented in Table 2. In both cases quantification was
based on the external standard method.
No significant differences between the results obtained with the present
method CY) and the comparison method (x)were obtained when determined by
ANOVA methodology. followed by Fisher's PLSD test. (Differences were
consideredsignificant for p < 0.01). Regression lines between the two methods
were y = 0.996 x + 0.108. (r = 0.997) and y = 1.01 x - 0.042. (r = 0.993) for
infant formulaeand breast milk. respectively.
CONCLUSION
An accurate and precise method to quantifytaurine in infant formulae and
breast milk is presented.
Despite the complexity of the matrix, the sample pre-treatment is simple
and this approach only requires a basic HPLC system without extra reagent
pumps, mixing manifolds, reaction coils, etc. which is an obvious advantage of
precolumn derivatisation relatively to post column derivatisation.' However. it
requires rigorous control of the OPA-reagent and reaction time in order to
obtain the high degreeof reproducibitily shown here.
The chromatographicrun time of 5 minutes is comparablewith the lowest
reported time for fluorimetric determinations'' and less than the run time
reported for UV determinations.'
ACKNOWLEDGEMENT
This work received financial support from Junta Nacional de Investigaqlo
Cientifica Project0 PBIC/C/TPR/ 2564/95.
REFERENCES
1. I. Zelikovic. R. W. Chesney. "Taurine", in New protectiveRoles for
Selected Nutrients. Alan R. Liss. Inc.. pag. 253-294. (1989).
Downloadedby[UniversityofWestFlorida]at06:1103October2014
1278 FERREIRA ET AL.
2. G. E. Gaull. Pediatrics. 83.133442. (1989)
3. R. W. Chesney. Ped. Res.. 22. 755-759 (1987).
4. First Report of the Scientific Committee for Food related with the
caracteristics of the infant formulae based on cow's milk. Commission of
the European Communities. Luxemburg. 14th sene. EUR 8752 (1983).
5. A. A. Stampfli. 0.Ballevre, L. B. Fay. J. Chromatogr.. 617, 197-203
(1985).
6. B. R. Larsen. D. S. Grosso. S. Y. Chang. J. Chromatogr.Scienc.. 18, 233-
236 (1980).
7. T. Hirai. H. Ohyama. R. Kido. Anal. Biochem.. 163,339-342 (1987).
8. T. Yokoyama. T. Kinoshita. J. Chroaatogr.. 106. 212-218 (1991).
9. T. Sakai. T. Nagasawa. (1992) J. Chromatogr.. 114. 155-157 (1992).
10.N. Masuoka. K. Yao. M. Kinuta. J. Ohta, M. Wakimoto, T. Ubuka. J.
Chromatogr. B. 660. 31-35 (1994).
11. C. J. Waterfield. J. Chromatogr. B, 657. 37-45 (1991) .
12.Z. L. Chen, G. Xu. K. Specht. R. J. Yang, S. W. She, Anal. Chim. Acta,
296. 249-253 (1994) .
Received August 6, 1996
Accepted August 26. 1996
Manuscript 4217
Downloadedby[UniversityofWestFlorida]at06:1103October2014

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Ferreira1997

  • 1. This article was downloaded by: [University of West Florida] On: 03 October 2014, At: 06:11 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Journal of Liquid Chromatography & Related Technologies Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljlc20 Development of An HPLC-UV Method for Determination of Taurine in Infant Formulae and Breast Milk I. M. P. L. V. O. Ferreira a , M. V. Nunes a , E. Mendes a , F. Remião b & M. A. Ferreira a a CEQUP b Laboratório de Bromatologia Laboratório de Toxicologia Faculdade de Farmácia, Universidade do Porto Rua Aníbal Cunha , 164 4050, Porto, Portugal Published online: 23 Sep 2006. To cite this article: I. M. P. L. V. O. Ferreira , M. V. Nunes , E. Mendes , F. Remião & M. A. Ferreira (1997) Development of An HPLC-UV Method for Determination of Taurine in Infant Formulae and Breast Milk, Journal of Liquid Chromatography & Related Technologies, 20:8, 1269-1278, DOI: 10.1080/10826079708010975 To link to this article: http://dx.doi.org/10.1080/10826079708010975 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the
  • 2. Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions Downloadedby[UniversityofWestFlorida]at06:1103October2014
  • 3. J. LIQ. CHROM. & REL. TECHNOL., 20(8), 1269-1278 (1997) DEVELOPMENT OF AN HPLC-UV METHOD FOR DETERMINATION OF TAURINE IN INFANT FORMULAE AND BREAST MILK I. M. P.L. V. 0.Ferreira,t M. V. Nunes, E. Mendes, F. Remiilo,I M. A. Ferreirat ~CEQUP tLaboratoriode Bromatologia Laboratbriode Toxicologia Faculdade de Farmacia, Universidade do Porto Rua Anibal Cunha 164 4050 Porto, Portugal ABSTRACT A rapid and accurate hgh performance liquid chromato- graphic (HPLC)procedurewas proposed for routine and selective determination of taurine in infant formulas and breast milk. The sample preparation was simple, involving protein removal and filtration operations. Afterwards, taurine was derivatised with 0- phthalaldehyde/2-mercaptoethanol prior to injection onto a reversed phase column CI8 (SloODS2). Isocratic elution was carried out using 0.05 M phosphate buffer pH 5.3/ methanol (60:40) mixture. The effluentwas monitored by a UV detector at 350 nm. A linear relationship was found between peak area and a concentration range of 1-70 pg/mL. The detection limit was 0.3 pg/mL. Effective separation and quantification was achieved in under six minutes. No interference of other amino acids was observed. 1269 Copyright 8 1997by Marcel Dekker. Inc. Downloadedby[UniversityofWestFlorida]at06:1103October2014
  • 4. 1270 FERREIRA ET AL. Extensive validation of the proposed method was carried out both by the standard additions method and by comparison with a fluorimetric method of known accuracy. The precision was better than 1.4%. INTRODUCTION Taurine. 2-aminoethanesulphonic acid. is a sulphur-containing p-amino acid that is not incorporated into proteins. However, it takes part in biochemical reactions of major importance, such as conjugation with bile acids to form bile salts essential for fat absorption, cell membrane stabilization, antioxidation. detoxification. osmoregulation, neuromodulation, and brain and retinal development.’ Considerable evidence has accumulated that neonates and infants, who have a very limited ability to synthesizetaurine, are especially prone to develop taurine deficiency and that they depend on an external taurine supply.’ Taurine concentrations in milk are very variable, depending on species. Wlule breast milk is an excellent source of taurine in the developing infant. cow’s milk has a very low taurine content. Nowadays the supplementation of infant formulae with taurine in concentrations similar to those found in human milk is therefore re~ommended.~.‘ Many methods for measuring taurine in several matrices viz. biological samples, such as tissue homogenates, urine, and serum have been published, including gas chromatography’ and HPLC analysis of diferent amino acid derivatives.6-” One of the most sensitive and commonly used derivatising agent techniques is the use of o-phthaldehyde / mercaptoethanol, which, originates a fluorescent adduct that enables the fluorimetric determination of taurine.6*1’ Other HPLC fluorimetric determinations in similar matrices include derivatising reaction with hamine*and fluore~camine.~ It is also possible to use UV absorbance detection at 350 nm with different derivatisation techniques. includmg o-phthaldehyde / mercaptoethanol’ and dinitrofluorobenzene.‘~,’~Interestingly. research work developed by Chen at aI.l2is distinguished from the rest since one of the matrices used was human milk. Downloadedby[UniversityofWestFlorida]at06:1103October2014
  • 5. TAURINE IN W A N T FORMSJLAE AND BREAST MILK 1271 Some of these methods require time-consuming pre-treatment of the samples. in order to eliminate interfering substances or complicated derivatising treatment of the samplebefore assay. This paper describesa rapid and accurate method for the determination of taurine in infant formulae and breast milk. Although, the sample matrix is of complex nature, the pre-treatment applied is simple and the method is suitable for rapid routine assays of a large number of samples and uses a UV detector which is much more common in control laboratories than the fluorimetric detector. MATERIALS Apparatus The chromatographic analysis was carried out in a Gilson, high performance liquid Chromatograph (Gilson Medical Electronics, Villiers le Bel, France) equipped with a type305 pump, a type 302 pump and a type 7125 Rheodyne Injector with a 20 p1 loop. A Gilson 118, variable long wave ultra violet detector was also used. The chromatographic separation was achieved with a Spherisorb Slo ODs2Chromatographiccolumn, 10 pm. The integrator used was a Gilson 712 HPLC System Controller Software. A Gilson 121 Spectrofluorometerwas used for detection of the fluorescent adduct. Reagents and Standards Taurine and o-phthaldehydewere obtained from Sigma Chemical Co. L- Amino acids kit was also from Sigma (Kit No. LAA-21). Sulphosalicylicacid, boric acid, sodium hydroxide and mercaptoethanol,all p.a., were obtained from Merck. Methanol (LiCrosolv)was Merck "gradient grade". Water used for chromatography possessed a resistance greater than 15 Ma. and was filtered through a membrane of 0.45 pm porosity which was subsequentlydegassed. Downloadedby[UniversityofWestFlorida]at06:1103October2014
  • 6. 1272 FERREIRA ET AL. Sample Preparation After homogenisation. 5.0 g of infant formulae were dissolved in 40 mL of warm (40°C) water. 5.0 mL of the infant formulae solution or breast milk were added to 5.0 mL of sulphosalicylic acid solution 0.2 M. mixed thorougly. and allowed to stand for 10 min. All samples were filtered through W42 paper and thereafter. through 0.2 pm filter paper. The use of sulphosalicylic acid as a protein precipitating agent has been shown to result in a higher YOrecovery of taurine.” Denvatisation Procedure Taurine was derivatised with o-phthalaldehyde (OPA, 40 mg, 0.8 mL absolute ethanol) and 2-mercaptcethanol (4Opl) in 0.5 M borate buffer (10 mL, 3.1 g boric acid in 90 mL water. adjusted to pH 10.4with 5M NaOH. and made up to 100 mL)6prior to injection onto a reversed phase column CIS(SloODS2). The derivatizing solution was prepared daily and filtered through a 0.20 pm filter before use. A 100 pL volume of standard or sample solution (after preparation) was placed in an eppendorf and 100 pl of derivatizing reagent were added. The derivatization reaction was allowed to proceed for exactly 1.5 min, at which time an aliquot was injected into the HPLC. Chromatography The HPLC elution required a mixture of two solvents. Solvent A. 0.05 M phosphate buffer pH 5.3. and solvent B. methanol. The two solvents were filtered and degassedbefore use. Isocratic elution was carried out at a flow rate of 1.5 mL/min, using 60% solvent A combined with 40 YOsolvent B. The injection volume was 20 p1 and the chromatographic analysis was conducted at ambient temperature. Taurine concentration was determined at an absorbance of 350 nm. which is the maximum wavelength on the excitation spectrum of OPA-derivatized amino acids. Taurine peak was identified by coelution with a standard and by comparison of the retention time. Downloadedby[UniversityofWestFlorida]at06:1103October2014
  • 7. TAURINE IN INFANT FORMULAE AND BREASTMILK 1273 Figure 1. Standard curves for taurine. Taurine was derivatised and chromatographed by the method described in the text. Each point represents the average of triplicate determinations. (a) Sensitivityof 0.06 AUFS, detection limite 1 p g / d . (b) Sensitivity of 0.03AUFS, detection liniite 0.3 pg/mL. RESULTS Analytical Curve and Detection Limit Under the assay conditions described, a linear relationship between the concentrationof taurine and the UV absorbance at 350 nm was obtained. This linearity was mantained over the concentration range 3-70 pg/mL with a sensitivityof 0.06AUFS and 1-10 pg/mL with a sensitivity of 0.03 AUFS. The detection limit value was calculated as the concentrationcorrespondingto three times the standard deviation of the background noise and was 1 pg/mL and 0.3 pg/mL, respectively. The calibrationcurvesfor taurine are ilustratedin Fig. 1. Downloadedby[UniversityofWestFlorida]at06:1103October2014
  • 8. 1274 FERREIRAET AL. .... Figure 2. Typical chromatogram obtained for taurine extracted from infant formulae. Retention time of taurine 4.51. Triplicate determinations were made on each of the calibration standards, the peak area values (arbitrary units) that were plotted are average values. The relative percent average deviationsof triplicates were less than 3 YOin all cases. Validity of the Method The repeatability of the derivatization procedure was examined. A standard taurine solution was derivatized and chromatographed six times. When reaction and chromatographic conditions were carefidly optimized, the standard deviationwas 0.02 (n=6 and concentrationof taurine 20 pg/mL). Downloadedby[UniversityofWestFlorida]at06:1103October2014
  • 9. T A W IN INFANTFORMULAE AND BREASTMILK 1275 Table 1 Recovery of Taurine from Spiked Infant Formulae &mL pg/mL Standard CV% Recovery Added Found’ Deviation YO 2.00 2.03 M.05 2.46 101.5 Sample 1 4.00 3.92 M.08 2.04 98.0 6.00 5.82 M.09 1.54 97.0 2.00 1.99 34.03 1.51 99.5 Sample9 4.00 4.10 k0.07 1.71 102.5 6.00 5.73 34.06 1.05 95.5 “Meanvalue found for 3 assays for each studiedconcentrations. The interference of other amino acids was examined nest. For this purpose, different amino acids were injected after derivatization with OPA. They included L-Alanine, L-Valine, L-Leucine, L-Isoleucine. L-Proline. L- Phenylalanine,L- Tryptophan, L- Methionine, Glycine, L-Serine, L-Threonine, L-Tyrosine, L-Asparagine. L-Glutamine. L-Aspartic acid. L-Glutamic acid. L- Lysine, L-Arginine, L-Histidine. Different retention times were obtained for each amino acid. Fig. 2 shows a typical chromatograph of taurine extracted from infant formulae. Peaks corresponding to several amino acids were detected in the chromatograms obtained for infant formulae determinations, but as shown, these peaks were well separated from those of taurine under the conditions used. A similar behaviourwas observedwith regards to breast milk. The precision of the analytical method was evaluated by measuring the peak chromatographicarea 10times on the same sample. The relative standard deviation (RSD) ranged between 0.7 and 1.4 YO(concentration of taurine in infant formula 42.6 and 28.6 mg of taurineA00 g of infant formula. respectively). Recovery studies were carried out on two different samples of infant formulae, sample 1 with high concentration of taurine, and sample 9 with very low concentration of taurine. The samples were analysed in triplicate before and after the addition of known amounts of taurine and they were spiked with Downloadedby[UniversityofWestFlorida]at06:1103October2014
  • 10. 1276 FERREIRA ET AL. Table 2 Comparison Between Taurine Determined in Infant Formulae and Breast Milk by HPLC-UV and a Comparison Method of Known Accuracy' Taurine Determined (mg/100 g of Infant Formula) Sample No. HpLC-UV(y) Comparison method (x) 1 2 3 4 5 6 7 8 9 10 39.4 f0.7b 34.4 f 1.3b 28.6 f0.3b 39.0 f 0.9b 38.0 f 0.2b 44.7 f0.7b 32.7 *0.9b 30.2 f l.lb 1.O1 f 0.08" 3.99 f O.2lC 40.6 f 0.5 32.7 f0.2 27.7 *0.7 10.3k 0.5 38.8 f 0.6 43.1 +0.3 32.9 f0.1 31.0 f 0.9 0.80 ? 0.04 4.32 k0.11 Taurine Determined (mg/100 mL of Breast Milk) 11 4.13 f0.06b 12 4.25 kO.Ogb 13 2.28 f0.1lb 14 2.18 f0.09b 4.23 f 0.10 4.32 f0.05 2.43 f0.08 2.08 f0.12 aValuesare expressed as mean fstandard deviation of three determinations. Sensitivity of 0.06 AUFS. "Sensitivityof 0.03 AUFS. Samples 1 to 10are from infant formulae, samples 11 to 14 are from breast milk. b three different concentrations of taurine. The results are listed in Table 1. The addition of taurine to the infant formulae was made before the protein removal. Thus, this procedure proved the effectiveness of the extraction step and the accuracy of the proposed method. Recovery values were between 95.5 and 102.5%. which confirm no interference effects due to matrix composition. Downloadedby[UniversityofWestFlorida]at06:1103October2014
  • 11. TAURINJ?IN INFANT FORMULAE AND BREAST MILK 1277 In order to validate the accuracy of the analytical method and because there was no certified reference material available, not only the standard addition method was made. but also a comparison with a method of known accuracy." The results of these determinations of taurine in infant formulae and breast milk are presented in Table 2. In both cases quantification was based on the external standard method. No significant differences between the results obtained with the present method CY) and the comparison method (x)were obtained when determined by ANOVA methodology. followed by Fisher's PLSD test. (Differences were consideredsignificant for p < 0.01). Regression lines between the two methods were y = 0.996 x + 0.108. (r = 0.997) and y = 1.01 x - 0.042. (r = 0.993) for infant formulaeand breast milk. respectively. CONCLUSION An accurate and precise method to quantifytaurine in infant formulae and breast milk is presented. Despite the complexity of the matrix, the sample pre-treatment is simple and this approach only requires a basic HPLC system without extra reagent pumps, mixing manifolds, reaction coils, etc. which is an obvious advantage of precolumn derivatisation relatively to post column derivatisation.' However. it requires rigorous control of the OPA-reagent and reaction time in order to obtain the high degreeof reproducibitily shown here. The chromatographicrun time of 5 minutes is comparablewith the lowest reported time for fluorimetric determinations'' and less than the run time reported for UV determinations.' ACKNOWLEDGEMENT This work received financial support from Junta Nacional de Investigaqlo Cientifica Project0 PBIC/C/TPR/ 2564/95. REFERENCES 1. I. Zelikovic. R. W. Chesney. "Taurine", in New protectiveRoles for Selected Nutrients. Alan R. Liss. Inc.. pag. 253-294. (1989). Downloadedby[UniversityofWestFlorida]at06:1103October2014
  • 12. 1278 FERREIRA ET AL. 2. G. E. Gaull. Pediatrics. 83.133442. (1989) 3. R. W. Chesney. Ped. Res.. 22. 755-759 (1987). 4. First Report of the Scientific Committee for Food related with the caracteristics of the infant formulae based on cow's milk. Commission of the European Communities. Luxemburg. 14th sene. EUR 8752 (1983). 5. A. A. Stampfli. 0.Ballevre, L. B. Fay. J. Chromatogr.. 617, 197-203 (1985). 6. B. R. Larsen. D. S. Grosso. S. Y. Chang. J. Chromatogr.Scienc.. 18, 233- 236 (1980). 7. T. Hirai. H. Ohyama. R. Kido. Anal. Biochem.. 163,339-342 (1987). 8. T. Yokoyama. T. Kinoshita. J. Chroaatogr.. 106. 212-218 (1991). 9. T. Sakai. T. Nagasawa. (1992) J. Chromatogr.. 114. 155-157 (1992). 10.N. Masuoka. K. Yao. M. Kinuta. J. Ohta, M. Wakimoto, T. Ubuka. J. Chromatogr. B. 660. 31-35 (1994). 11. C. J. Waterfield. J. Chromatogr. B, 657. 37-45 (1991) . 12.Z. L. Chen, G. Xu. K. Specht. R. J. Yang, S. W. She, Anal. Chim. Acta, 296. 249-253 (1994) . Received August 6, 1996 Accepted August 26. 1996 Manuscript 4217 Downloadedby[UniversityofWestFlorida]at06:1103October2014