Analytica Chimica Acta 562 (2006) 185189

HPLC-ESI-MS analysis of Vitamin B12 in food products and

in multivitamins-multimineral tablets
Xubiao Luo a,b , Bo Chen b , Li Ding a , Fei Tang a , Shouzhuo Yao a,b,
a State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, PR China
b Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research, Ministry of Education,

Hunan Normal University, Changsha 410081, PR China

Received 17 November 2005; received in revised form 21 January 2006; accepted 23 January 2006
Available online 28 February 2006

Abstract
Methods for the determination of Vitamin B12 remain limited due to their low sensitivity and poor selectivity. In the present work, a simple and
sensitive HPLC-ESI-MS method for determining Vitamin B12 in food products and in multivitamin-multimineral tablets was developed. Vitamin
B12 was extracted from food products with 50 mM sodium acetate buffer (pH 4.0) in the presence of sodium cyanide. Total Vitamin B12 content
in food product can be obtained by efficient enzymatic hydrolysis to release the bound Vitamin B12 . Vitamin B12 was quantified with ginsenoside
Re as internal standard (I.S.) after their separations on a C18 column with a gradient of mobile phase made of water and acetonitrile. MS with
SIR mode at m/z 930.8 was used for Vitamin B12 quantification. The calibration graphs plotted with five concentrations of Vitamin B12 was linear
with a regression coefficient r2 = 0.9994. The intra-assay R.S.D. and the inter-assay R.S.D. were 2.6% (n = 5) and 3.5% (n = 6), respectively. The
recoveries evaluated at spiking three different concentrations on fortified products were above 93%. The method has been applied successfully in
the determination of Vitamin B12 in fortified food products and multivitamin-multimineral tablets.
2006 Elsevier B.V. All rights reserved.

Keywords: Vitamin B12 ; Fortified products; Liquid chromatography-lectrospray ionization-mass spectrometry; Food analysis

1. Introduction Various analytical methods including microbiological assay

Vitamin B12 (cyanocobalamin), a tetrapyrrole complex which
contains a cobalt atom in the molecule, is an important species
in human physiology. The deficiency of Vitamin B12 may cause
pernicious anemia and neuropathy [1]. The main source of vita-
mins for human beings is from food. However, the amount of
vitamins required for the normal development and maintenance
of body functions are usually insufficiency only depending on
the human diet. To prevent such deficiency diseases, people are
advised to intake adequate amount of Vitamin B12 daily. Vitamin
tablet and fortified foods is preferred worldwide. Considering the
great number of vitamin-taking people and the variety of com-
mercially available vitamin products, fast, simple, and sensitive
analytical methods for products quality control of Vitamin B12
are desired.
the sensitivity is rather low. Chemiluminescence [10,11] and
Corresponding author. Tel.: +86 731 8865515; fax: +86 731 8865515.
E-mail addresses: dr-chenpo@vip.sina.com (B. Chen),
shouzhuoyao@126.com (S. Yao).
[24], radioisotope dilution assay [57], spectrophotometry
[8,9], chemiluminescence [10,11], atomic absorption spectrom-
etry [12,13], electrochemical analysis [1416], matrix-assisted
laser desorption/ionization (MALDI) time-of-flight mass spec-
trometry (TOFMS) [1719] or plasma desorption mass spec-
trometry [20], multinuclear solid-state NMR analysis [21], cap-
illary electrophoresis [22,23], and high performance liquid chro-
matography [2439] have been proposed for the detection of
Vitamin B12 . The official method for Vitamin B12 determination
is commonly accomplished by microbiological assay using Lac-
tobacillus leishmanii as test organism [2], despite this method
is tedious, time-consuming, poor precision, and relatively low
specificity. Radioisotopic dilution method [57] is simple and
rapid, but expensive due to the requirement of radio-labeled
cyanocobalamin and intrinsic factor of high purity. Spectropho-
tometry [8,9] is unsuitable for the complex sample matrix and
atomic absorption spectrometry [12,13] are based on indirect
determination of cobalt, however, the applicability of these

0003-2670/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2006.01.073
186 X. Luo et al. / Analytica Chimica Acta 562 (2006) 185189
methods for quantification of Vitamin B12 in real sample is
somewhat limited since these methods cannot distinguish the
free and bonded form of cobalt in Vitamin B12 . Capillary elec-
trophoresis and chromatographic methods with various modes
for determining of Vitamin B12 have been proposed, includ-
ing UV or visible photometry [22,2434], atomic absorption
[35], and ICPMS [23,36]. Unfortunately, these methods are
not appropriate for the determination of Vitamin B12 in food
product due to the low content (320 ng/g) of Vitamin B12 .
Li et al. [37] proposed a method for determination of Vita-
min B12 by HPLC with fluorescence detection. The method
was not able to handle more complex samples such as pro-
cessed foods, where Vitamin B12 quantification was challenging.
Pakin et al. [38] proposed Vitamin B12 should be derivatized
into a fluorescent complex first, and then analyzed by HPLC
with fluorescence detection. The purification step and enrich-
ment were necessary when dealing with complex sample. Heudi
et al. [39] reported a narrow bore LC approach that was com-
bined immunoaffinity purification. The immunoaffinity column
is, however, not commercially available. Moreover, enrichment
and use of a narrow bore LC column were also necessary. Addi-
tionally, this procedure was not suitable for routine analyses
of large numbers of samples due to the complicated pretreat-
ment procedures. This shortcoming can be overcome by LC/MS
to achieve better selectivity and sensitivity for quantification
of Vitamin B12 in which a conventional C18 column is uti-
lized.
The introduction of atmospheric pressure ionization tech-
nique greatly expanded the number of compounds and matri-
ces that can be analyzed by HPLC-ESI-MS. However, the
analysis of large molecules, such as Vitamin B12 (molecular
mass, 1355 Da), is limited due to their low ionization effi-
ciency. To our knowledge, the report on the analysis of Vita-
min B12 using HPLC-ESI-MS has not been found. The pur-
pose of the present study was to establish a sensitive HPLC-
ESI-MS method for the determination of Vitamin B12 in
food products and multivitamin-multimineral tablets. By sim-
plifying the sample preparation process and applying high
cone voltage, the sensitive HPLC-ESI-MS method has been
obtained.
2. Experimental
2.1. Chemicals
HPLC-grade methanol and acetonitrile were purchased from
Tedia Company, Inc. (Fairfield, OH, USA). Vitamin B12 was
obtained from Sigma (St. Louis, MO, USA). Ginsenoside Re
was purchased from National Research Center for CRMS (Bei-
jing, China). Sodium cyanide was purchased from Tianheng
Chemical Reagents Company (Changsha, China). Deionized
water via an ultra-water system from Millipore (Milford Bed-
ford, MA, USA) was used throughout. The enzymes, pepsin
(EC3.4.23.1, EC3.4.23.1, Sigma, catalogue No. P 7125) and -
amylase (EC3.2.1.1, Sigma, catalogue No. A 2771), were used.
All other reagents used were analytical grade.
2.2. Samples
21-Super-Vita multivitamin-multimineral tablets were manu-
factured by MIN SHENG Pharmaceutical Co. Ltd. (Hangzhou,
Zhejiang, China). Shanghai Jinwei multivitamin-multimineral
tablets (21) were manufactured by SHANGHAI Pharmaceuti-
cal Factory (Nanchang, Jiangxi, China). Nestle pure full cream
milk powder, Nestle high calciumhigh iron milk powder, Nestle
lactogen starter formula milk powder, Yili child milk pow-
der, and Yili follow-up milk powder were purchased at local
sources.
2.3. Solutions preparation
The stock solution of Vitamin B12 (125 g/mL) was prepared
by dissolving 12.5 mg of Vitamin B12 in 100 mL of deionized
water. I.S. solution of ginsenoside Re (40 g/mL) was also pre-
pared in deionized water. A set of standard solutions containing
Vitamin B12 in the concentration range of 6150 ng/mL and
0.1 g/mL of I.S. were prepared. The solutions were stored at
4 C, the storage life is at least 3 months.
2.4. Free Vitamin B12 extraction
The sample was treated according to references [38,39]
with some modifications. The sample (1530 g, depending on
the content of Vitamin B12 in the food product) was accu-
rately weighed in a flask. Forty milliliters of 50 mM sodium
acetate buffer (pH 4.0), 1 mL of sodium cyanide (1%) and
0.25 g of -amylase were added under agitation and the solu-
tion was incubated at 42 C for 30 min. The pH value of the
solution was adjusted to 4.8 using sodium hydroxide solutions
and then heated at 100 C for 35 min under nitrogen steam
reflux and agitation. After cooling to room temperature, the
solution was quantitatively transferred in a 50 mL of volumet-
ric flask, added 125 L of internal standard solution, filled
to the mark with deionized water. The resulting solution was
shaken fully, centrifuged at 8000 g for 10 min. The super-
natant was filtered through a 0.45 m membrane filter before
injection.
For the premix samples, two tablets (1.6 g) were accurately
weighed in a flask. Forty milliliters of 50 mM sodium acetate
buffer, and 1 mL of sodium cyanide (1%) was added under
agitation and the solution was incubated at 42 C for 30 min.
After cooling to room temperature, the solution was handled as
described above.
2.5. Total Vitamin B12 extraction
The sample (1530 g, depending on the content of Vitamin
B12 in the food product) was accurately weighed in a flask.
Forty milliliters of 50 mM sodium acetate buffer (pH 4.0), 1 mL
of sodium cyanide (1%), 0.25 g of -amylase, and 1 g of pepsin
were added under agitation and the solution was incubated at
37 C for 3 h. After cooling the solution was handled as described
in Section 2.4.
 X. Luo et al. / Analytica Chimica Acta 562 (2006) 185189
2.6. HPLC-ESI-MS
Waters HPLC system (Waters Corporation, Milford, MA,
USA) was equipped with in-line degasser AF, 600 pump, 600
controller, and 2777C sample manager, and connected to Micro-
mass ZQ 4000 electrospray mass spectrometer (Manchester,
U.K.). Masslynx V 3.4 software (Micromass) was used for
data acquisition and processing. A XTerraTM MS C18 column
(3.9 mm 150 mm, 5 m, Milford, MA, USA) was used. The
separations were performed at room temperature. A linear gra-
dient eluent of A (water) and B (acetonitrile) was used. The
gradient elution was programmed as follows: 05 min, 515%
B; 510 min, 1530% B; 1011 min, 5% B; 1115 min, 5% B.
The solvent flow rate was set at 1 mL/min while the injection vol-
ume was 50 L. Only 0.2 mL/min portion of column eluent was
delivered into the mass spectrometry by solvent splitting. The
ESI-MS spectra were acquired in the positive ion mode. Nitro-
gen was used as both desolvation gas at a flow rate of 350 L/h
and cone gas at a flow rate 50 L/h. The desolvation temperature
was set at 350 C. The ionization source was working at 105 C.
Capillary and cone voltages were 4000 and 180 V, respectively.
Quantifier ions used for Vitamin B12 and ginsenoside Re were
m/z 930.8 and 969.9, respectively.
3. Results and discussion
3.1. Optimization of separation conditions
Isocratic elution using methanol/water, methanol/water con-
taining 0.1% formic acid, acetonitrile/water, and acetoni-
trile/water containing 0.1% formic acid as eluent were com-
pared. Ion suppression was found when formic acid was added
in the mobile phase. Moreover, the signal-noise ratio (S/N) could
be improved using acetonitrile as mobile phase. Therefore, the
mobile phase system of acetonitrile/water was selected.
The effect of C18 , CN, and C8 on the separation of Vita-
min B12 was explored. The appropriate retention time has been
obtained with C18 column. For optimization of eluent composi-
tion, different gradient elution conditions were also investigated.
Under gradient elution conditions described in Section 2.6, good
resolution between Vitamin B12 and I.S. were achieved.
A suitable internal standard is necessary in quantitatively
determining Vitamin B12 . The ideal internal should be the iso-
topically labeled Vitamin B12 . However, commercial stable-
isotope-labeled Vitamin B12 was not available. Therefore, a
number of compounds, such as phenol, hydroquinone, benzoic
acid, p-phthalic acid, caffeine, and ginsenoside Re were inves-
tigated. Similar retention time and good response of ESI-MS
have been obtained with ginsenoside Re as the internal stan-
dard. Ginsenoside Re was selected as the internal standard in
the subsequent experiments.
3.2. Cone voltage and quantiers ions optimization
The influence of cone voltage, capillary voltage, desolvation
gas, and desolvation temperature were investigated carefully.
The maximum value of S/N of Vitamin B12 was obtained with
187
4000 V of capillary voltage, 350 L/h of desolvation gas flow rate,
and 350 C of desolvation temperature. The experiments indi-
cate the effects of the parameters, capillary voltage, desolvation
gas flow rate, and desolvation temperature exceeded above val-
ues, were negligible. Whereas, the effect of the cone voltage was
obvious on the determination of Vitamin B12 .
Optimizing desorption/ionization processes in order to
enhance analyte ion yields and reduce fragmentation are popular
in mass spectrometry. Kinsel et al. [18] reported MALDI of Vita-
min B12 yielded low abundances of the protonated molecule in
TOF mass spectra. In the present work, dispersed pseudomolec-
ular ions with low abundance, which is m/z 679.0, 690, 697.5,
701.5, 1356.1, and 1378.0 at cone voltage 30 V, were observed
in the ESI mass spectrometry. Six pseudomolecular ions are
corresponding to [M + 2H]2+ , [M + H + Na]2+ , [M + H + K]2+ ,
[M + 2Na]2+ , [M + H]+ , and [M + Na]+ , respectively. Therefore,
it is difficult to obtain high sensitivity for determining Vitamin
B12 . Our purpose is to optimize high abundance of characteristic
fragmentation ion in order to enhance sensitivity. First, the ESI
mass spectra of Vitamin B12 with different ESI cone voltages
were investigated. Vitamin B12 is a fragile molecule with labile
peripheral groups. The mechanism of fragment ion formation
is mainly collisional activation in source collision induced dis-
sociation (CID). Thus, the axial side-chain groups were firstly
dissociated. At 50 V of cone voltage, the following fragment
ions, m/z 1015.9, 989.8, 930.8, 912.7, 657.9, 585.1, 525.0, 663.6,
have been obtained. The m/z 1015.9 represents to [M + H-base-
sugar-PO3 ]+ , the m/z 989.8 corresponds to [M + H-base-sugar-
PO3 -CN]+ (also reported in TOF mass spectra [1719]), and
the m/z 930.8 corresponds to [M + H-base-sugar-PO3 -CN-Co]+ .
Whereas, the m/z 912.7, 657.9, 585.1, 525.0, 663.6 fragment
ions remain unknown, and further detailed investigations are
required.
In order to select quantifier ions, the peak area of the charac-
teristic ions of Vitamin B12 at their optimization cone voltages
has been measured. The results are illustrated in Fig. 1. Fig. 1
indicates that the optimal peak area of m/z 930.8 has reached
Fig. 1. Peak area average values (n = 3) of characteristic ions from Vitamin B12
obtained at difference optimal cone voltages: (1) m/z 525.0 (70 V), (2) m/z 585.1
(80 V), (3) m/z 657.9 (60 V), (4) m/z 679.0 (40 V), (5) m/z 690.0 (30 V), (6) m/z
697.5 (40 V), (7) m/z 701.5 (40 V), (8) m/z 912.7 (180 V), (9) m/z 930.8 (180 V),
(10) m/z 1015.9 (130 V), (11) m/z 1356.1 (70 V), (12) m/z 1378.0 (100 V), (13)
m/z 1394.1 (100 V).
188 X. Luo et al. / Analytica Chimica Acta 562 (2006) 185189
at 180 V of cone voltage. The peak area of m/z 930.8 is almost
two times higher than the peak area of m/z 1015.9 at 130 V or
that of m/z 690.0 at 30 V, and is more than 15 times higher than
the peak area of m/z 1356.1 at 70 V or that of m/z 1394.1 at
100 V. At the same time, an interesting phenomenon has been
found that the baseline noise at low cone voltage is almost two
times higher than that in high cone voltage. High value of peak
area and low baseline noise result in two order of magnitude
lower detection limit of m/z 930.8 at 180 V than these of m/z
1356.1 at 70 V or m/z 1394.1 at 100 V. Therefore, m/z 930.8
was selected as quantifier ions, and cone voltage was set at
180 V.
3.3. Method validation
3.3.1. Specicity
Specificity, described as the ability of a method to discrim-
inate the analyte from potential interfering substances, was
evaluated by preparing the placebo and blank milk powder.
Solutions of a placebo and blank milk powder were simulta-
neously prepared according to the sample extraction procedure
without internal standard and injected. The representative chro-
matogram is shown in Figs. 2 and 3. Figs. 2 and 3 show that
no obvious interference was observed on the determination of
Vitamin B12 and ginsenoside Re. The result expresses excel-
lent specificity of the method for the determination of Vitamin
B12 .
3.3.2. Linearity and LOD
Under the optimum analysis conditions, linearity was stud-
ied over the concentration range of 6150 ng/mL for Vitamin milk powder.
B12 in the presence of 0.1 g/mL internal standard. The peak
normalization ratios of Vitamin B12 to the internal standard
Fig. 2. The HPLC-SIR-MS chromatograms of SIR + 930.8 of placebo (A) and
SIR + 930.8 of multivitamin-multimineral tablets (B), SIR + 969.9 of placebo
(C) and SIR + 969.9 of multivitamin-multimineral tablets (D). Chromatographic
conditions are described in Section 2.6.
Fig. 3. The HPLC-SIR-MS chromatograms of SIR + 930.8 of blank milk powder
(A) and SIR + 930.8 of Nestle pure full cream milk powder, 1.8 g Vitamin
B12 /100 g (B), SIR + 969.9 of placebo (C) and SIR + 969.9 of Nestle pure full
cream milk powder (D). Chromatographic conditions are described in Section
2.6.
were plotted versus the nominal concentrations of the calibration
standards. The linearity curves were defined by the following
equations: y = (0.057 0.001)x + (0.0059 0.001), r2 = 0.9994,
where y is the ratio of peak normalization and x is the concentra-
tion expressed in ng/mL (n = 5). The limits of detection defined
as signal-to-noise ratio of 3 was about 2 ng/g for Vitamin B12 in
3.3.3. Precision
The precision of the intra-daily (five times per day) and
inter-daily (twice a day for three consecutive days) analytical
determinations was evaluated based on the calculation of the
R.S.D. of the ratio of peak area values. The obtained R.S.D.
values of intra-day (2.6%) and inter-day (3.5%) showed that the
precision of the proposed method was satisfying.
3.3.4. Recovery
Recovery experiments were performed using the standard
addition method. Three difference contents of Vitamin B12
were spiked to the blank milk powder. Samples were treated
as described in the procedure for sample extraction. The results
were shown in Table 1. The recoveries of Vitamin B12 were
above 93%. The results showed the recovery of the proposed
method was satisfying.
Table 1
Recovery for Vitamin B12 in blank milk powder (n = 3)
Spiked Found Recovery (%) R.S.D.
concentration (ng/g) concentration (ng/g)
8 7.5 93.8 3.3
16 15.3 95.6 2.8
32 31.2 97.5 3.5
 X. Luo et al. / Analytica Chimica Acta 562 (2006) 185189 189

Table 2
Determination of Vitamin B12 content in milk powder product and multivitamin-multimineral sample

Samplea Labeled content of Vitamin B12 (ng/g) Free Vitamin B12 (ng/g)b Total Vitamin B12 (ng/g)b

Nestle pure full cream milk powder (20050928) 18 15.3 17.6

Nestle high calciumhigh iron milk powder (20050523) 16 12.6 15.5
Nestle lactogen milk powder (20050702) 15 13.1 14.2
Yili child milk powder (20050907) 10 8.9 9.3
Yili follow-up milk powder (20050829) 12 10.4 12.1
21-Super-Vita tablets (20040621) 625 637.5 630.5
21-Super-Vita tablets (20041005) 625 640.3 641.2
21-Super-Vita tablets (20041013) 625 625.0 625.5
Shanghai Jinwei tablets (20041109) 625 639.5 640.1
Shanghai Jinwei tablets (20041219) 625 610.7 615.3
Shanghai Jinwei tablets (20050220) 625 643.5 641.2
a Different production date.
b n = 5.

3.4. Sample analysis [6] F. Watanabe, Y. Nakano, E. Stupperich, K. Ushikoshi, S. Ushikoshi, I.

The present method was applied to determine Vitamin B12 in
food products and multivitamin-multimineral tablets manufac-
tured by two factories. Results are collected in Table 2. The
contents of Vitamin B12 in food products and multivitamin-
multimineral are in agreement with the value declared by the
manufacturers. The results show that the proposed method can
be used in quality control of Vitamin B12 products.
4. Conclusions
A simple and sensitive HPLC-ESI-MS analytical method for
Vitamin B12 quality control during production and at the end
of shelf life has been developed. The sensitivity of Vitamin
B12 determination is significantly enhanced by selecting high
cone voltage without micro bore LC column. Low LOD (2 ng/g)
of Vitamin B12 in milk powder was reached without laborious
clean-up, enrichment, and derivation procedures in comparison
with those methods previously mentioned. This novel method
can be applied in the throughput analysis of Vitamin B12 in food
products and multivitamin-multimineral tablets.
Acknowledgements
This work was financially supported by the High-Tech
Research and Development (863) Program of Ministry of
Science and Technology of PR China (2001BA804A21,
2001BA804A39) and the Natural Science Foundation of Hunan
Province (03JJY1002).
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