Accepted Manuscript Analytical methods Rapid determination of vitamin D3 in milk-based infant formulas by liquid chromatography-tandem mass spectrometry Byung-Man Kwak, In-Seek Jeong, Moon-Seok Lee, Jang-Hyuk Ahn, Jong-Su Park PII: DOI: Reference:
S0308-8146(14)00854-1 http://dx.doi.org/10.1016/j.foodchem.2014.05.137 FOCH 15921
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
9 November 2012 10 February 2014 27 May 2014
Please cite this article as: Kwak, B-M., Jeong, I-S., Lee, M-S., Ahn, J-H., Park, J-S., Rapid determination of vitamin D3 in milk-based infant formulas by liquid chromatography-tandem mass spectrometry, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.05.137
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1
Rapid determination of vitamin D3 in milk-
2
based infant formulas by liquid
3
chromatography-tandem mass spectrometry
4 5 6 7
Byung-Man Kwak, In-Seek Jeong, Moon-Seok Lee, Jang-Hyuk Ahn*, Jong-Su Park
8
Food Safety Center, Research and Development Institute, Namyang Dairy Co., Ltd,
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Sejong 314-914, Republic of Korea
10 11 12 13
*To whom correspondence should be addressed:
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Tel.: +82-44-856-0381; fax: +82-44-857-7933.
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E-mail address: [email protected]
16 17 18
1
19
ABSTRACT
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A rapid and simple sample preparation method for vitamin D3 (cholecalciferol) was developed
21
for emulsified dairy products such as milk-based infant formulas. A sample was mixed in a 50
22
mL centrifuge tube with the same amount of water and isopropyl alcohol to achieve chemical
23
extraction. Ammonium sulfate was used to induce phase separation. No-heating saponification
24
was performed in the sample tube by adding KOH, NaCl, and NH3. Vitamin D3 was then
25
separated and quantified using liquid chromatography-tandem mass spectrometry. The results
26
for added recovery tests were in the range 93.11–110.65%, with relative standard deviations
27
between 2.66% and 2.93%. The results, compared to those obtained using a certified reference
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material (SRM 1849a), were within the range of the certificated values. This method could be
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implemented in many laboratories that require time and labor saving.
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Keywords: Vitamin D3; Cholecalciferol; No-heating saponification; dSPE; LC-MS/MS; Milk-
33
based infant formula.
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1. Introduction
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Cholecalciferol, vitamin D3, is a fat-soluble vitamin that is essential for maintaining normal
38
calcium metabolism (Holick, 2004). Vitamin D3 is a white, crystalline compound that is stable
39
against heat and oxidation in mild alkaline or acidic solutions (Friedrich, 1988). Vitamin D has
40
many isomers. Vitamin D3 is synthesized in human skin on exposure to ultraviolet (UV)
41
radiation from sunlight, or it can be obtained from food. Plants synthesize ergosterol, which is
42
converted to vitamin D2 (ergocalciferol) by UV light (Holick, 2003). The dehydrogenation of
43
cholesterol generates 7-dehydrocholesterol in vivo (Mercer & Glover, 1961; Robbins,
44
Thompson, Kaplanis, & Shortino, 1964), and 7-dehydrocholesterol is converted to vitamin D3
45
by UV irradiation in the range 295–315 nm (Rajakumar, Greenspan, Thomas, & Holick, 2007).
46
These sterol-like compounds have similar structures and can be analyzed by similar
47
instrumental analytical procedures (Sullivan & Carpenter, 1993).
48
Vitamin D is naturally found in trace amounts in some foods. Foods containing vitamin D
49
include some fatty fishes (mackerel, salmon, sardines), fish liver oils, and eggs from hens that
50
have been fed vitamin D. Among dairy products, processed milk products and infant formulas
51
are fortified with vitamin D. In the USA, infant formulas generally contain 400 IU (10 µg) per
52
quart, but cheese and yogurt are not generally vitamin D supplemented (Food and Nutrition
3
53
Board, Institute of Medicine, 1999). Internationally, most vitamin D containing foods are
54
enriched with vitamin D3.
55
Sample preparation of emulsified foods such as infant formulas is not easy, and the official
56
methods require much time and proficiency for accurate and precise analysis. The official
57
methods for determining vitamin D3 and D2 are the International Dairy Federation (IDF) method
58
for dried skimmed milk and the European Standard (EN) method for general commodities
59
(European Standard EN 12821, 2009; International Standard ISO 14892│IDF 177, 2002). These
60
methods require accuracy, precision, and stability to be established using liquid chromatography
61
(LC)-UV spectrometry. However, current analytical trends tend to use LC-tandem mass
62
spectrometry (LC-MS/MS) for accurate determination of target compounds (Soler & Pico,
63
2007).
64
An additional official method, the Association of Official Analytical Chemists (AOAC)
65
method 2011.13, which is used to evaluate infant formulas and adult nutritionals, makes use of
66
LC-MS/MS in selected reaction monitoring (AOAC Official Method 2011.13, 2012; Gilliland,
67
Black, Denison, Seipelt, & Dowell, 2012). In order to perform more accurate analyses of target
68
materials, in recent years, multiple reaction monitoring (MRM) mode has generally been used in
69
LC-MS/MS instrumental analysis. A triple quadruple mass spectrometer with MRM mode
70
provides sensitive detection for identification of an analyte (Kind & Fiehn, 2010). The fact that
4
71
the MRM mode is capable of evaluating two or more product ions (qualitatively and
72
quantitatively) from one precursor ion, is a major factor in increasing MS accuracy.
73
Other methods have been developed to increase the analysis quality using LC-MS/MS MRM
74
mode. These methods were developed for the determination of vitamin D in bovine milk
75
(Trenerry, Plozza, Caridi, & Murphy, 2011), meat (Jakobsen, Clausen, Leth, & Ovesen, 2004),
76
human serum (Guo, Talyor, Singh, & Soldin, 2006), and blood spots (Eyles et al., 2009). In the
77
present study, we used the methods mentioned above. However, both the official and non-
78
official methods, using saponification by heating and liquid–liquid partitioning with a separation
79
funnel, make the experimental times very long.
80
In a previous study, a rapid method was developed for the determination of cholesterol in milk-
81
containing emulsified foods, including infant formulas (Ahn et al., 2012). Considering the
82
structural similarity of cholesterol and vitamin D3 , a similar rapid and simple analytical method
83
was applied to vitamin D3 in this study. LC-MS/MS was used in order to separate and detect
84
vitamin D3 from the sample solution of infant formula matrix. Optimum conditions were
85
established for no-heating saponification, dispersive solid phase extraction (dSPE) for the
86
cleaning phase, and LC-MS/MS for accurate determination of vitamin D3.
87
2. Materials and methods
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5
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2.1. Samples and reagents
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The infant formulas used in this study were purchased from a local market and stored at 4 °C.
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A certified reference material (CRM), infant formula SRM 1849a (NIST, Gaithersburg, MD,
92
USA), was used in the recovery tests to develop the method. The amount of vitamin D3 in SRM
93
1849a was 111 ± 17 µg/kg.
94
Reagent-grade ammonium sulfate ((NH4)2SO4) anhydrous, and sodium chloride (NaCl) were
95
purchased from Sigma-Aldrich (St. Louis, MO, USA). Potassium hydroxide (KOH) was
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purchased from Junsei Chemical (Tokyo, Japan). Isopropyl alcohol (IPA), used as the extraction
97
solvent, was purchased from Fisher Scientific (Hampton, NH, USA). The dSPE sorbent,
98
Discovery® DSC-NH2 (NH2 ), was purchased from Supelco (Bellefonte, PA, USA). Ultrapure
99
water was obtained using a Banstead Diamond TII system (Dubuque, IA, USA). The distilled
100
water had a resistance of 18.0 MΩ.
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Vitamin D3 (cholecalciferol) was purchased from the US Pharmacopeial Convention (USP,
102
Washington, DC, USA) for use as the reference standard material. A stock solution of 100
103
ng/mL vitamin D3 was dissolved in IPA, and diluted to the required concentrations of 1, 5, 10,
104
50, and 100 ng/mL to make the standard working solutions.
105
2.2. Sample pretreatment
106
The sample pretreatment process has been previously described (Ahn et al., 2012). Infant
6
107
formulas and SRM 1849a were tested for the determination of vitamin D3. Sample amount for
108
0.5, 1.0, and 2.0 g was placed into respective three 50 mL centrifuge tubes and dissolved in 10
109
mL of distilled water at room temperature. After the addition of 10 mL of IPA as the extraction
110
solvent, the solutions were covered with a screw cap and vigorously mixed for 1 min using a
111
vortex mixer at maximum speed. Subsequently, 4.0 g of (NH4)2SO4 anhydrous were added as a
112
precipitating agent for the separation of IPA from the aqueous solution, and immediately
113
vortexed for 30 s. The tube was then centrifuged for 5 min at 4000 rpm at 4 °C.
114
The upper lipidic layer (6 mL) was transferred into a 15 mL centrifuge tube, and 150 mg of
115
KOH, 150 mg of NH3, and 2.4 g of NaCl were added. The tube was vigorously mixed for 1 min
116
and then kept for 1, 5, 10, 20, and 30 min at room temperature. This step was necessary to
117
determinate the optimum no-heating saponification time. After the addition of 6 mL of distilled
118
water, the solutions were covered with a screw cap and vigorously mixed for 1 min using a
119
vortex mixer at maximum speed. Subsequently, the tube was centrifuged for 5 min at 4000 rpm
120
at 4 °C. A 3 mL aliquot of the solution was concentrated to 0.5 mL (six-fold) by nitrogen
121
evaporation. Finally, the solution was filtered using a 0.45 µm polyvinylidene difluoride (PVDF)
122
filter, and transferred to a vial for autosampling.
123 124
2.3. Instrumental conditions
7
125
The instrumental analysis conditions were determined by analogy with previous reports using
126
LC-MS/MS (Capote, Jimenez, Granados, & Castro, 2007; Gilliland, Black, Denison, Seipelt, &
127
Dowell, 2012; Trenerry, Plozza, Caridi, & Murphy, 2011). An Agilent 1200 HPLC system
128
(Agilent, Palo Alto, CA, USA) equipped with an Xbridge C18 (3.5 µm, 2.1 × 150 mm) reverse-
129
phase column (Waters, Milford, MA, USA) and a 6410 triple quadrupole LC/MS tandem MS
130
system were used for analysis of vitamin D3. The final LC-MS/MS conditions used for the
131
analysis are shown in Table 1.
132 133
2.4. Method validation
134
The method was validated based on a recovery tests, linearity, limit of detection (LOD), limit
135
of quantification (LOQ), and method detection limit (MDL). The recovery tests were carried out
136
by SRM 1849a and spiking with standard in the infant formula sample. The linearity of the
137
calibration curve was evaluated by the average coefficient of determination (r2). It was
138
calculated using five consecutive points of standard solution. The LOD and LOQ were
139
determined by diluting a vitamin D3 standard working solutions to obtain signal to noise ratios
140
of ~3:1 for LOD and ~10:1 for LOQ. The vitamin D3 standard was weighed (~ 0.1 g) and
141
dissolved in 100 mL of IPA. MDL was determined by multiplying the solvent volume (mL) of
142
the LOD and dividing by the sample amount (g).
8
143
3. Results and discussion
144 145
3.1. Applications for sample pretreatment
146
The sample preparation method previously developed for cholesterol, which is the major
147
unsaponificable component in infant formulas, was successfully used for vitamin D3 (Ahn et al.,
148
2012). Since vitamin D3 is a sterol-like compound, it was expected that during the pretreatment
149
process, it would behave like cholesterol. The core applications used in this study were
150
published as IDF International Methods for Fatty Acids Determination (International Standard
151
ISO 14156│IDF 172, 2001; International Standard ISO 15884│IDF 182, 2002; International
152
Standard ISO 23065│IDF 211, 2009). In these methods, highly concentrated KOH solution
153
was used for alkaline hydrolysis of fat globules and saponification technique without heating
154
was used. These procedures involved sampling small amounts of extracted fats. For rapid
155
pretreatment, when optimizing the appropriate sample weight, microextraction techniques were
156
chosen in order to use smaller solvent volumes. Thus, the dimensions of the liquid–liquid
157
partitioning instrumentation were reduced from a funnel shaker to a 50 mL tube. It was also
158
determined whether, in contrast to the AOAC method (AOAC Official Method 2011.13, 2012;
159
Gilliland, Black, Denison, Seipelt, & Dowell, 2012), the optimum no-heating saponification
160
time could be reduced to 30 min.
9
161
3.2. Optimum sample weight
162
To determine the optimum sample weight for extracting vitamin D3 using IPA 10 mL and water
163
10 mL, three sample weight was evaluated by using 0.5, 1.0, and 2.0 g. Each sample was taken
164
into the 50 mL centrifuge tube, respectively. The test result for the determining optimum sample
165
weight was shown in Fig. 1. When using 0.5 g of initial sample weight, the recovery for each
166
sample was in the acceptable range of 88.13–106.74%. In the case of the standard SRM 1849a,
167
recovery was satisfactory when using a sample of weight 1 g. However, the infant formula
168
samples showed low and rapidly decreasing recoveries for sample weights greater than 1 g. The
169
recoveries of SRM 1849a and infant formulas increased as the sample weight decreased. When
170
the sample weight was below 0.5 g, the amounts of vitamin D3 in some infant formula products
171
were below the detection limit. Based on these results, 0.5 g of sample weight was considered to
172
be the optimum sample weight and was adopted for further tests.
173 174
3.3. Determination of optimum no-heating saponification time
175
Typically, in the published official methods, saponification for the determination of vitamin D3
176
was performed for 20–45 min at 70–100 °C (AOAC Official Method 2011.13, 2012; European
177
Standard EN 12821, 2009; Gilliland, Black, Denison, Seipelt, & Dowell, 2012; International
178
Standard ISO 14892│IDF 177, 2002). In order to determine the optimum saponification time
10
179
without heating, extracted infant formula samples and SRM 1849a were kept for 1, 5, 10, 20,
180
and 30 min after shaking for 1 min using a vortex mixer. The results for saponification time
181
optimization are shown in Fig. 2. Within a saponification time range of 1–30 min, there were no
182
significant differences (p < 0.05) among the tested values for the three infant formula samples
183
(infant formula sample 1, 2, and 4). However, infant formula sample 3 and SRM 1849a showed
184
different results for 1 min and 5 min. When the saponification time was 1 min, the tested value
185
of SRM 1849a was out of the range for its certificated value. Infant formula sample 3 also
186
displayed a tested value 2.5 times higher than that obtained for a saponification time over 5 min.
187
Based on these results, 5 min of no-heating saponification time was evaluated to be the
188
optimum condition for 0.5 g of infant formula.
189 190
3.4. Conditions for LC-MS/MS with MRM mode
191
Mass spectrometry is used as a standard technique for the analytical investigation of molecules
192
and complex mixtures. It is important in determining the elemental composition of a molecule
193
and in gaining partial structural insights, using MS fragmentations (Kind & Fiehn, 2010). Like
194
other emulsified processed foods, powdered infant formulas have very complex food matrixes.
195
In addition, since vitamin D3 in infant formulas is present in extremely small quantities, precise
196
analytical instruments are essential when using MS analysis.
11
197
In this study, the establishment of MS conditions was conducted by following the guidelines
198
for the determination of vitamin D3 specified by the European Community (EC) and the Codex
199
Alimentarius Commission (CAC) (Commission decision of 12 August 2002, 2002; Codex
200
guideline, 2007). The details for MS/MS instrumentation are shown in Table 1;
201
chromatographic and MS spectral results are shown in Fig. 3. The precursor ion for vitamin D3
202
corresponded to 385 m/z, the product qualitative ions were 259 and 159 m/z, respectively, and
203
the quantitative ion was 107 m/z. These results for ion fragments were consistent with previous
204
results reported for bovine milk (Trenerry, Plozza, Caridi, & Murphy, 2011). Under these
205
conditions, by comparison with the standard deviation of the relative response values, the results
206
for SRM 1849a and infant formula samples were within the acceptable range.
207
The relative response for 259 m/z per 107 m/z (qualitative and quantitative ions, respectively)
208
showed a deviation of less than 30%, whereas 159 m/z per 107 m/z showed a deviation of less
209
than 20%. For a more accurate analysis, collision energies for the product ions 259, 159, and
210
107 m/z were set to 10, 20, and 24, respectively. With this analysis, another fragment, with 367
211
m/z was detected, and was revealed to be largest product ion in our study. However, that
212
fragment was not stable at various collision energies among all the samples, and even the
213
standard material, although mass 367 (m/z) was the largest product ion for fragmentation of 385
214
m/z.; 367 m/z was considered to be an inappropriate qualitative ion for MS/MS analysis. In this
12
215
study, LC-MS/MS analysis of vitamin D3 in infant formulas was successfully conducted using
216
the same experimental conditions previously applied to bovine milk (Trenerry, Plozza, Caridi, &
217
Murphy, 2011), although infant formula milk has a more complex matrix. Moreover, sample
218
solutions produced using the sample preparation method of this study were successfully
219
analyzed by LC-MS/MS.
220 221
3.5 Validation and monitoring tests
222
Four infant formula samples and SRM 1849a were analyzed using the method developed in
223
this study. Following the AOAC guidelines for single laboratory validation of chemical methods
224
(AOAC International, 2002), sample pretreatments were repeated three times for each sample.
225
Test results and recoveries are shown in Table 2. The tested value of 119.50 ± 3.26 µg/kg for
226
SRM 1849a was within the range of the certified value. The tested values for infant formula
227
samples were in the range 61.36–121.02 µg/kg, falling below 120% of the labeled values. In the
228
spiking tests, the recoveries ranged from 93.11% to 110.65%, and the relative standard deviation
229
values ranged from 2.66 to 2.93%. In contrast to the official methods, this procedure required a
230
smaller amount of sample, and a sample pretreatment time of 20 min.
231
The LOD and LOQ values for vitamin D3 were 0.396 µg/kg and 1.307 µg/kg, respectively. The
232
MDL value was 0.84 µg/kg for infant formula; this determined value was adequate, since the
13
233
typical vitamin D contents (vitamin D3 and D2) in infant formulas are in range 60–100 µg/kg
234
(United States Department of Agriculture, 2011). The LOQ and MDL values for vitamin D3 on
235
AOAC official method were 1.68 µg/kg and 0.6 µg/kg, respectively (AOAC Official Method
236
2011.13, 2012; Gilliland, Black, Denison, Seipelt, & Dowell, 2012). As above, our method was
237
possible to analyze the levels for µg/kg (ppb) such as AOAC method.
238
The linearity of the detector for vitamin D3 ranged from 1 to 100 ng/mL for five levels. The
239
tandem mass detector gave a linear response over the range of concentrations used, and a least-
240
squares linear regression analysis of the data provided excellent r2 values (>0.9999), which
241
indicated a good fit for the calibration function. Considering the above results, the method
242
developed in this study enables accurate analysis of vitamin D3, Importantly, the pretreatment
243
time was improved with respect to existing methods. A comparison between the AOAC official
244
method and the developed method is shown in Fig. 4. The developed method ensures reduced
245
experimental times and labor, which is particularly useful when dealing with multiple samples.
246 247
4. Conclusions
248 249
A rapid and accurate method for the determination of vitamin D3 in milk products such as
250
infant formulas was developed. The major characteristics of this procedure include small sample
14
251
amounts,
252
experiments. Although sample dimensions were reduced, the results showed excellent
253
recoveries. Consequently, the efficiency of the preparation time (1 h and 25 minutes per each
254
sample when processing 6 samples at the same time) and costs could be dramatically decreased
255
when using this analytical method. The MRM mode for LC-MS/MS was found to be optimal for
256
instrumental analysis. The precursor ion was 385 m/z, the product qualitative ions were 259 and
257
159 m/z, and the product quantitative ion was 107 m/z. The recovery test successfully recorded
258
93.11–110.65% recoveries. Moreover, the result of vitamin D3 content using CRM was in the
259
range of the certificated value (SRM 1849a, NIST). The developed method based on LC-
260
MS/MS in MRM mode, following the described sample preparation, could be an accurate tool
261
that could replace the official methods when time and labor need to be reduced.
liquid–liquid separation, no-heating saponification, dSPE, and small-scale
262 263
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337 338 339
Figure legends
340
19
341
Fig. 1. Comparison of vitamin D3 contents in recovery tests to determine the optimum sample
342
amounts of SRM 1849a and infant formula.
343
Fig. 2. Comparison of vitamin D3 contents for test values to determine the optimum no-heating
344
saponification time for SRM 1849a and infant formulas.
345
Fig. 3. Liquid chromatography-tandem mass spectrometry chromatograms of vitamin D3 by the
346
total ion current and multiple reaction monitoring mode, with relative response ratios; Vitamin
347
D3 standard (a), SRM 1849a (b), and Infant formula sample (c).
348
Fig. 4. Sample pretreatment flow-charts displaying times for the determination of vitamin D3 in
349
infant formulas comparing the Association of Official Analytical Chemists method with
350
developed method.
351 352
Table legends
353 354
Table 1
355
Liquid chromatography-tandem mass spectrometry operating conditions for determination of
356
vitamin D3.
357 358
Table 2
20
359
Recovery and monitoring tests for vitamin D3 in certified reference material (SRM 1849a) and
360
infant formulas using no-heating saponification method with liquid chromatography-tandem
361
mass spectrometry analysis.
362
21
363
364 365
22
366 367
23
368 369
24
370 371
25
372
Table 1
373
Liquid chromatography-tandem mass spectrometry operating conditions for determination of
374
vitamin D3.
375
(a) LC Parameter
Condition
Column
Xbridge C18 3.5 µm, 2.1 × 150 mm (Waters)
Detector
MS/MS A : 5 mM Ammonium formate in Water B : 5 mM Ammonium formate in MeOH ※ Gradient
Mobile phase
Time (min)
%A
%B
Flow rate (mL/min)
Comment
0
6
94
0.2
Vitamin D3 elution
20
6
94
0.2
21
1
99
0.2
65
1
99
0.2
66
6
94
0.2
Faster equilibration
70
6
94
0.2
for next run
Column temperature
40 ℃
Running time
70 min
Injection volume
10 uL
376 377
26
Post-elution column wash
378
(b) MS/MS Parameter
Condition
Ion source
ESI (Electro spray ionization)
Polarity
Positive
Nebulizer gas
N2
Nebulizer pressure
36 psi
Gas flow
11 L/min
Ion spray voltage
3500 V
Source temp.
300 ℃
Resolution
Q1(unit) Q3(unit)
Scan mode
MRM (Multiple reation monitoring)
MRM condition Retention Time(min) 10.7
Precursor Compound ion
D3
(m/z)
Dwell Fragmentor Collision (ms)
(V)
Energy(V)
385
107 Quantitative 200
115
24
385
159 Qualitative
200
115
22
385
259 Qualitative
200
115
10
(m/z) Vitamin
Product ion
379 380
27
381
Table 2
382
Recovery and monitoring tests for vitamin D3 in certified reference material (SRM 1849a) and
383
infant formulas using no-heating saponification method with liquid chromatography-tandem
384
mass spectrometry analysis. Tested Valuea
Samples
(µg/kg)
SRM 1849ab
RSD (%)
119.50 ± 3.26
2.73
Spikedc
150.44 ± 3.64
2.72
Blank
77.95 ± 3.95
5.07
Spikedc
119.01± 1.78
2.68
Blank
61.36 ± 1.10
1.79
Spikedc
212.86 ± 3.63
2.93
Infant formula sample 1
Recovery (%)
107.65
93.11
Infant formula sample 2
93.95
Infant formula sample 3
102.86 Blank
104.93 ± 0.95
0.91
Spikedc
255.43 ± 4.76
2.66
Infant formula sample 4
110.65 Blank
121.02 ± 0.72
385 386
a
The values are mean ±S.D of 3 replications.
b
The certificated value of SRM 1849a was 111 ± 17 µg/kg.
387
c
The spiked levels of infant formula was 100 µg/kg.
388 389
28
0.59
390 391
Highlights
392 393
* No-heating saponification and dSPE clean-up was applied to extract trace vitamin D3 from
394
milk-based infant formula.
395
*Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for accurate
396
quantitative analysis.
397
*The result of recovery test using certified reference material showed 93.11-110.65%.
398
29
S0308-8146(14)00854-1 http://dx.doi.org/10.1016/j.foodchem.2014.05.137 FOCH 15921
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
9 November 2012 10 February 2014 27 May 2014
Please cite this article as: Kwak, B-M., Jeong, I-S., Lee, M-S., Ahn, J-H., Park, J-S., Rapid determination of vitamin D3 in milk-based infant formulas by liquid chromatography-tandem mass spectrometry, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.05.137
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Rapid determination of vitamin D3 in milk-
2
based infant formulas by liquid
3
chromatography-tandem mass spectrometry
4 5 6 7
Byung-Man Kwak, In-Seek Jeong, Moon-Seok Lee, Jang-Hyuk Ahn*, Jong-Su Park
8
Food Safety Center, Research and Development Institute, Namyang Dairy Co., Ltd,
9
Sejong 314-914, Republic of Korea
10 11 12 13
*To whom correspondence should be addressed:
14
Tel.: +82-44-856-0381; fax: +82-44-857-7933.
15
E-mail address: [email protected]
16 17 18
1
19
ABSTRACT
20
A rapid and simple sample preparation method for vitamin D3 (cholecalciferol) was developed
21
for emulsified dairy products such as milk-based infant formulas. A sample was mixed in a 50
22
mL centrifuge tube with the same amount of water and isopropyl alcohol to achieve chemical
23
extraction. Ammonium sulfate was used to induce phase separation. No-heating saponification
24
was performed in the sample tube by adding KOH, NaCl, and NH3. Vitamin D3 was then
25
separated and quantified using liquid chromatography-tandem mass spectrometry. The results
26
for added recovery tests were in the range 93.11–110.65%, with relative standard deviations
27
between 2.66% and 2.93%. The results, compared to those obtained using a certified reference
28
material (SRM 1849a), were within the range of the certificated values. This method could be
29
implemented in many laboratories that require time and labor saving.
30 31 32
Keywords: Vitamin D3; Cholecalciferol; No-heating saponification; dSPE; LC-MS/MS; Milk-
33
based infant formula.
34 35
2
36
1. Introduction
37
Cholecalciferol, vitamin D3, is a fat-soluble vitamin that is essential for maintaining normal
38
calcium metabolism (Holick, 2004). Vitamin D3 is a white, crystalline compound that is stable
39
against heat and oxidation in mild alkaline or acidic solutions (Friedrich, 1988). Vitamin D has
40
many isomers. Vitamin D3 is synthesized in human skin on exposure to ultraviolet (UV)
41
radiation from sunlight, or it can be obtained from food. Plants synthesize ergosterol, which is
42
converted to vitamin D2 (ergocalciferol) by UV light (Holick, 2003). The dehydrogenation of
43
cholesterol generates 7-dehydrocholesterol in vivo (Mercer & Glover, 1961; Robbins,
44
Thompson, Kaplanis, & Shortino, 1964), and 7-dehydrocholesterol is converted to vitamin D3
45
by UV irradiation in the range 295–315 nm (Rajakumar, Greenspan, Thomas, & Holick, 2007).
46
These sterol-like compounds have similar structures and can be analyzed by similar
47
instrumental analytical procedures (Sullivan & Carpenter, 1993).
48
Vitamin D is naturally found in trace amounts in some foods. Foods containing vitamin D
49
include some fatty fishes (mackerel, salmon, sardines), fish liver oils, and eggs from hens that
50
have been fed vitamin D. Among dairy products, processed milk products and infant formulas
51
are fortified with vitamin D. In the USA, infant formulas generally contain 400 IU (10 µg) per
52
quart, but cheese and yogurt are not generally vitamin D supplemented (Food and Nutrition
3
53
Board, Institute of Medicine, 1999). Internationally, most vitamin D containing foods are
54
enriched with vitamin D3.
55
Sample preparation of emulsified foods such as infant formulas is not easy, and the official
56
methods require much time and proficiency for accurate and precise analysis. The official
57
methods for determining vitamin D3 and D2 are the International Dairy Federation (IDF) method
58
for dried skimmed milk and the European Standard (EN) method for general commodities
59
(European Standard EN 12821, 2009; International Standard ISO 14892│IDF 177, 2002). These
60
methods require accuracy, precision, and stability to be established using liquid chromatography
61
(LC)-UV spectrometry. However, current analytical trends tend to use LC-tandem mass
62
spectrometry (LC-MS/MS) for accurate determination of target compounds (Soler & Pico,
63
2007).
64
An additional official method, the Association of Official Analytical Chemists (AOAC)
65
method 2011.13, which is used to evaluate infant formulas and adult nutritionals, makes use of
66
LC-MS/MS in selected reaction monitoring (AOAC Official Method 2011.13, 2012; Gilliland,
67
Black, Denison, Seipelt, & Dowell, 2012). In order to perform more accurate analyses of target
68
materials, in recent years, multiple reaction monitoring (MRM) mode has generally been used in
69
LC-MS/MS instrumental analysis. A triple quadruple mass spectrometer with MRM mode
70
provides sensitive detection for identification of an analyte (Kind & Fiehn, 2010). The fact that
4
71
the MRM mode is capable of evaluating two or more product ions (qualitatively and
72
quantitatively) from one precursor ion, is a major factor in increasing MS accuracy.
73
Other methods have been developed to increase the analysis quality using LC-MS/MS MRM
74
mode. These methods were developed for the determination of vitamin D in bovine milk
75
(Trenerry, Plozza, Caridi, & Murphy, 2011), meat (Jakobsen, Clausen, Leth, & Ovesen, 2004),
76
human serum (Guo, Talyor, Singh, & Soldin, 2006), and blood spots (Eyles et al., 2009). In the
77
present study, we used the methods mentioned above. However, both the official and non-
78
official methods, using saponification by heating and liquid–liquid partitioning with a separation
79
funnel, make the experimental times very long.
80
In a previous study, a rapid method was developed for the determination of cholesterol in milk-
81
containing emulsified foods, including infant formulas (Ahn et al., 2012). Considering the
82
structural similarity of cholesterol and vitamin D3 , a similar rapid and simple analytical method
83
was applied to vitamin D3 in this study. LC-MS/MS was used in order to separate and detect
84
vitamin D3 from the sample solution of infant formula matrix. Optimum conditions were
85
established for no-heating saponification, dispersive solid phase extraction (dSPE) for the
86
cleaning phase, and LC-MS/MS for accurate determination of vitamin D3.
87
2. Materials and methods
88
5
89
2.1. Samples and reagents
90
The infant formulas used in this study were purchased from a local market and stored at 4 °C.
91
A certified reference material (CRM), infant formula SRM 1849a (NIST, Gaithersburg, MD,
92
USA), was used in the recovery tests to develop the method. The amount of vitamin D3 in SRM
93
1849a was 111 ± 17 µg/kg.
94
Reagent-grade ammonium sulfate ((NH4)2SO4) anhydrous, and sodium chloride (NaCl) were
95
purchased from Sigma-Aldrich (St. Louis, MO, USA). Potassium hydroxide (KOH) was
96
purchased from Junsei Chemical (Tokyo, Japan). Isopropyl alcohol (IPA), used as the extraction
97
solvent, was purchased from Fisher Scientific (Hampton, NH, USA). The dSPE sorbent,
98
Discovery® DSC-NH2 (NH2 ), was purchased from Supelco (Bellefonte, PA, USA). Ultrapure
99
water was obtained using a Banstead Diamond TII system (Dubuque, IA, USA). The distilled
100
water had a resistance of 18.0 MΩ.
101
Vitamin D3 (cholecalciferol) was purchased from the US Pharmacopeial Convention (USP,
102
Washington, DC, USA) for use as the reference standard material. A stock solution of 100
103
ng/mL vitamin D3 was dissolved in IPA, and diluted to the required concentrations of 1, 5, 10,
104
50, and 100 ng/mL to make the standard working solutions.
105
2.2. Sample pretreatment
106
The sample pretreatment process has been previously described (Ahn et al., 2012). Infant
6
107
formulas and SRM 1849a were tested for the determination of vitamin D3. Sample amount for
108
0.5, 1.0, and 2.0 g was placed into respective three 50 mL centrifuge tubes and dissolved in 10
109
mL of distilled water at room temperature. After the addition of 10 mL of IPA as the extraction
110
solvent, the solutions were covered with a screw cap and vigorously mixed for 1 min using a
111
vortex mixer at maximum speed. Subsequently, 4.0 g of (NH4)2SO4 anhydrous were added as a
112
precipitating agent for the separation of IPA from the aqueous solution, and immediately
113
vortexed for 30 s. The tube was then centrifuged for 5 min at 4000 rpm at 4 °C.
114
The upper lipidic layer (6 mL) was transferred into a 15 mL centrifuge tube, and 150 mg of
115
KOH, 150 mg of NH3, and 2.4 g of NaCl were added. The tube was vigorously mixed for 1 min
116
and then kept for 1, 5, 10, 20, and 30 min at room temperature. This step was necessary to
117
determinate the optimum no-heating saponification time. After the addition of 6 mL of distilled
118
water, the solutions were covered with a screw cap and vigorously mixed for 1 min using a
119
vortex mixer at maximum speed. Subsequently, the tube was centrifuged for 5 min at 4000 rpm
120
at 4 °C. A 3 mL aliquot of the solution was concentrated to 0.5 mL (six-fold) by nitrogen
121
evaporation. Finally, the solution was filtered using a 0.45 µm polyvinylidene difluoride (PVDF)
122
filter, and transferred to a vial for autosampling.
123 124
2.3. Instrumental conditions
7
125
The instrumental analysis conditions were determined by analogy with previous reports using
126
LC-MS/MS (Capote, Jimenez, Granados, & Castro, 2007; Gilliland, Black, Denison, Seipelt, &
127
Dowell, 2012; Trenerry, Plozza, Caridi, & Murphy, 2011). An Agilent 1200 HPLC system
128
(Agilent, Palo Alto, CA, USA) equipped with an Xbridge C18 (3.5 µm, 2.1 × 150 mm) reverse-
129
phase column (Waters, Milford, MA, USA) and a 6410 triple quadrupole LC/MS tandem MS
130
system were used for analysis of vitamin D3. The final LC-MS/MS conditions used for the
131
analysis are shown in Table 1.
132 133
2.4. Method validation
134
The method was validated based on a recovery tests, linearity, limit of detection (LOD), limit
135
of quantification (LOQ), and method detection limit (MDL). The recovery tests were carried out
136
by SRM 1849a and spiking with standard in the infant formula sample. The linearity of the
137
calibration curve was evaluated by the average coefficient of determination (r2). It was
138
calculated using five consecutive points of standard solution. The LOD and LOQ were
139
determined by diluting a vitamin D3 standard working solutions to obtain signal to noise ratios
140
of ~3:1 for LOD and ~10:1 for LOQ. The vitamin D3 standard was weighed (~ 0.1 g) and
141
dissolved in 100 mL of IPA. MDL was determined by multiplying the solvent volume (mL) of
142
the LOD and dividing by the sample amount (g).
8
143
3. Results and discussion
144 145
3.1. Applications for sample pretreatment
146
The sample preparation method previously developed for cholesterol, which is the major
147
unsaponificable component in infant formulas, was successfully used for vitamin D3 (Ahn et al.,
148
2012). Since vitamin D3 is a sterol-like compound, it was expected that during the pretreatment
149
process, it would behave like cholesterol. The core applications used in this study were
150
published as IDF International Methods for Fatty Acids Determination (International Standard
151
ISO 14156│IDF 172, 2001; International Standard ISO 15884│IDF 182, 2002; International
152
Standard ISO 23065│IDF 211, 2009). In these methods, highly concentrated KOH solution
153
was used for alkaline hydrolysis of fat globules and saponification technique without heating
154
was used. These procedures involved sampling small amounts of extracted fats. For rapid
155
pretreatment, when optimizing the appropriate sample weight, microextraction techniques were
156
chosen in order to use smaller solvent volumes. Thus, the dimensions of the liquid–liquid
157
partitioning instrumentation were reduced from a funnel shaker to a 50 mL tube. It was also
158
determined whether, in contrast to the AOAC method (AOAC Official Method 2011.13, 2012;
159
Gilliland, Black, Denison, Seipelt, & Dowell, 2012), the optimum no-heating saponification
160
time could be reduced to 30 min.
9
161
3.2. Optimum sample weight
162
To determine the optimum sample weight for extracting vitamin D3 using IPA 10 mL and water
163
10 mL, three sample weight was evaluated by using 0.5, 1.0, and 2.0 g. Each sample was taken
164
into the 50 mL centrifuge tube, respectively. The test result for the determining optimum sample
165
weight was shown in Fig. 1. When using 0.5 g of initial sample weight, the recovery for each
166
sample was in the acceptable range of 88.13–106.74%. In the case of the standard SRM 1849a,
167
recovery was satisfactory when using a sample of weight 1 g. However, the infant formula
168
samples showed low and rapidly decreasing recoveries for sample weights greater than 1 g. The
169
recoveries of SRM 1849a and infant formulas increased as the sample weight decreased. When
170
the sample weight was below 0.5 g, the amounts of vitamin D3 in some infant formula products
171
were below the detection limit. Based on these results, 0.5 g of sample weight was considered to
172
be the optimum sample weight and was adopted for further tests.
173 174
3.3. Determination of optimum no-heating saponification time
175
Typically, in the published official methods, saponification for the determination of vitamin D3
176
was performed for 20–45 min at 70–100 °C (AOAC Official Method 2011.13, 2012; European
177
Standard EN 12821, 2009; Gilliland, Black, Denison, Seipelt, & Dowell, 2012; International
178
Standard ISO 14892│IDF 177, 2002). In order to determine the optimum saponification time
10
179
without heating, extracted infant formula samples and SRM 1849a were kept for 1, 5, 10, 20,
180
and 30 min after shaking for 1 min using a vortex mixer. The results for saponification time
181
optimization are shown in Fig. 2. Within a saponification time range of 1–30 min, there were no
182
significant differences (p < 0.05) among the tested values for the three infant formula samples
183
(infant formula sample 1, 2, and 4). However, infant formula sample 3 and SRM 1849a showed
184
different results for 1 min and 5 min. When the saponification time was 1 min, the tested value
185
of SRM 1849a was out of the range for its certificated value. Infant formula sample 3 also
186
displayed a tested value 2.5 times higher than that obtained for a saponification time over 5 min.
187
Based on these results, 5 min of no-heating saponification time was evaluated to be the
188
optimum condition for 0.5 g of infant formula.
189 190
3.4. Conditions for LC-MS/MS with MRM mode
191
Mass spectrometry is used as a standard technique for the analytical investigation of molecules
192
and complex mixtures. It is important in determining the elemental composition of a molecule
193
and in gaining partial structural insights, using MS fragmentations (Kind & Fiehn, 2010). Like
194
other emulsified processed foods, powdered infant formulas have very complex food matrixes.
195
In addition, since vitamin D3 in infant formulas is present in extremely small quantities, precise
196
analytical instruments are essential when using MS analysis.
11
197
In this study, the establishment of MS conditions was conducted by following the guidelines
198
for the determination of vitamin D3 specified by the European Community (EC) and the Codex
199
Alimentarius Commission (CAC) (Commission decision of 12 August 2002, 2002; Codex
200
guideline, 2007). The details for MS/MS instrumentation are shown in Table 1;
201
chromatographic and MS spectral results are shown in Fig. 3. The precursor ion for vitamin D3
202
corresponded to 385 m/z, the product qualitative ions were 259 and 159 m/z, respectively, and
203
the quantitative ion was 107 m/z. These results for ion fragments were consistent with previous
204
results reported for bovine milk (Trenerry, Plozza, Caridi, & Murphy, 2011). Under these
205
conditions, by comparison with the standard deviation of the relative response values, the results
206
for SRM 1849a and infant formula samples were within the acceptable range.
207
The relative response for 259 m/z per 107 m/z (qualitative and quantitative ions, respectively)
208
showed a deviation of less than 30%, whereas 159 m/z per 107 m/z showed a deviation of less
209
than 20%. For a more accurate analysis, collision energies for the product ions 259, 159, and
210
107 m/z were set to 10, 20, and 24, respectively. With this analysis, another fragment, with 367
211
m/z was detected, and was revealed to be largest product ion in our study. However, that
212
fragment was not stable at various collision energies among all the samples, and even the
213
standard material, although mass 367 (m/z) was the largest product ion for fragmentation of 385
214
m/z.; 367 m/z was considered to be an inappropriate qualitative ion for MS/MS analysis. In this
12
215
study, LC-MS/MS analysis of vitamin D3 in infant formulas was successfully conducted using
216
the same experimental conditions previously applied to bovine milk (Trenerry, Plozza, Caridi, &
217
Murphy, 2011), although infant formula milk has a more complex matrix. Moreover, sample
218
solutions produced using the sample preparation method of this study were successfully
219
analyzed by LC-MS/MS.
220 221
3.5 Validation and monitoring tests
222
Four infant formula samples and SRM 1849a were analyzed using the method developed in
223
this study. Following the AOAC guidelines for single laboratory validation of chemical methods
224
(AOAC International, 2002), sample pretreatments were repeated three times for each sample.
225
Test results and recoveries are shown in Table 2. The tested value of 119.50 ± 3.26 µg/kg for
226
SRM 1849a was within the range of the certified value. The tested values for infant formula
227
samples were in the range 61.36–121.02 µg/kg, falling below 120% of the labeled values. In the
228
spiking tests, the recoveries ranged from 93.11% to 110.65%, and the relative standard deviation
229
values ranged from 2.66 to 2.93%. In contrast to the official methods, this procedure required a
230
smaller amount of sample, and a sample pretreatment time of 20 min.
231
The LOD and LOQ values for vitamin D3 were 0.396 µg/kg and 1.307 µg/kg, respectively. The
232
MDL value was 0.84 µg/kg for infant formula; this determined value was adequate, since the
13
233
typical vitamin D contents (vitamin D3 and D2) in infant formulas are in range 60–100 µg/kg
234
(United States Department of Agriculture, 2011). The LOQ and MDL values for vitamin D3 on
235
AOAC official method were 1.68 µg/kg and 0.6 µg/kg, respectively (AOAC Official Method
236
2011.13, 2012; Gilliland, Black, Denison, Seipelt, & Dowell, 2012). As above, our method was
237
possible to analyze the levels for µg/kg (ppb) such as AOAC method.
238
The linearity of the detector for vitamin D3 ranged from 1 to 100 ng/mL for five levels. The
239
tandem mass detector gave a linear response over the range of concentrations used, and a least-
240
squares linear regression analysis of the data provided excellent r2 values (>0.9999), which
241
indicated a good fit for the calibration function. Considering the above results, the method
242
developed in this study enables accurate analysis of vitamin D3, Importantly, the pretreatment
243
time was improved with respect to existing methods. A comparison between the AOAC official
244
method and the developed method is shown in Fig. 4. The developed method ensures reduced
245
experimental times and labor, which is particularly useful when dealing with multiple samples.
246 247
4. Conclusions
248 249
A rapid and accurate method for the determination of vitamin D3 in milk products such as
250
infant formulas was developed. The major characteristics of this procedure include small sample
14
251
amounts,
252
experiments. Although sample dimensions were reduced, the results showed excellent
253
recoveries. Consequently, the efficiency of the preparation time (1 h and 25 minutes per each
254
sample when processing 6 samples at the same time) and costs could be dramatically decreased
255
when using this analytical method. The MRM mode for LC-MS/MS was found to be optimal for
256
instrumental analysis. The precursor ion was 385 m/z, the product qualitative ions were 259 and
257
159 m/z, and the product quantitative ion was 107 m/z. The recovery test successfully recorded
258
93.11–110.65% recoveries. Moreover, the result of vitamin D3 content using CRM was in the
259
range of the certificated value (SRM 1849a, NIST). The developed method based on LC-
260
MS/MS in MRM mode, following the described sample preparation, could be an accurate tool
261
that could replace the official methods when time and labor need to be reduced.
liquid–liquid separation, no-heating saponification, dSPE, and small-scale
262 263
References
264 265
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312
chromatography.
and omega-6
fatty acid content by gas-liquid
313
Jakobsen, J., Clausen, I., Leth, T., & Ovesen, L. (2004). A new method for the determination of
314
vitamin D3 and 25-hydroxyvitamin D3 in meat. Journal of Food Composition and
315
Analysis, 17, 777–787.
316 317
Kind, T., & Fiehn, O. (2010). Advances in structure elucidation of small molecules using mass spectrometry. Bioanalytical Reviews, 2, 23–60.
318
Mercer, E.I., & Glover, J. (1961). Sterol metabolism. 6. The interconversion of cholesterol, 7-
319
dehydrocholesterol and lathosterol in the rat. Biochemical Journal, 80, 552–556.
320
Rajakumar, K., Greenspan, S.L., Thomas, S.B., & Holick, M.F. (2007). SOLAR ultraviolet
321
radiation and vitamin D: a historical perspective. American Journal of Public Health, 97,
322
1746–1754.
18
323
Robbins, W.E., Thompson, M.J., Kaplanis, J.N., & Shortino, T.J. (1964). Conversion of
324
cholesterol to 7-dehydrocholesterol in aseptically reared german cockroaches. Steroids,
325
4, 635–644.
326
Soler, C., & Pico, Y. (2007). Recent trends in liquid chromatography-tandem mass spectrometry
327
to determine pesticides and their metabolites in food. Trends in Analytical Chemistry, 26,
328
103–115.
329 330
Sullivan, D. M., & Carpenter, D. E. (1993). Methods of analysis for nutrition labeling: Chapter 5 Lipid Analysis (pp. 85–103). Arlington, VA, USA: AOAC International.
331
Trenerry, V. C., Plozza, T., Caridi, D., & Murphy, S. (2011). The determination of vitamin D3 in
332
bovine milk by liquid chromatography mass spectrometry. Food Chemistry, 125, 1314–
333
1319.
334
United States Department of Agriculture. (2011). USDA National Nutrient Database for
335
Standard Reference, Release 24. USDA Agricultural Research Service, Beltsville, MD,
336
USA.
337 338 339
Figure legends
340
19
341
Fig. 1. Comparison of vitamin D3 contents in recovery tests to determine the optimum sample
342
amounts of SRM 1849a and infant formula.
343
Fig. 2. Comparison of vitamin D3 contents for test values to determine the optimum no-heating
344
saponification time for SRM 1849a and infant formulas.
345
Fig. 3. Liquid chromatography-tandem mass spectrometry chromatograms of vitamin D3 by the
346
total ion current and multiple reaction monitoring mode, with relative response ratios; Vitamin
347
D3 standard (a), SRM 1849a (b), and Infant formula sample (c).
348
Fig. 4. Sample pretreatment flow-charts displaying times for the determination of vitamin D3 in
349
infant formulas comparing the Association of Official Analytical Chemists method with
350
developed method.
351 352
Table legends
353 354
Table 1
355
Liquid chromatography-tandem mass spectrometry operating conditions for determination of
356
vitamin D3.
357 358
Table 2
20
359
Recovery and monitoring tests for vitamin D3 in certified reference material (SRM 1849a) and
360
infant formulas using no-heating saponification method with liquid chromatography-tandem
361
mass spectrometry analysis.
362
21
363
364 365
22
366 367
23
368 369
24
370 371
25
372
Table 1
373
Liquid chromatography-tandem mass spectrometry operating conditions for determination of
374
vitamin D3.
375
(a) LC Parameter
Condition
Column
Xbridge C18 3.5 µm, 2.1 × 150 mm (Waters)
Detector
MS/MS A : 5 mM Ammonium formate in Water B : 5 mM Ammonium formate in MeOH ※ Gradient
Mobile phase
Time (min)
%A
%B
Flow rate (mL/min)
Comment
0
6
94
0.2
Vitamin D3 elution
20
6
94
0.2
21
1
99
0.2
65
1
99
0.2
66
6
94
0.2
Faster equilibration
70
6
94
0.2
for next run
Column temperature
40 ℃
Running time
70 min
Injection volume
10 uL
376 377
26
Post-elution column wash
378
(b) MS/MS Parameter
Condition
Ion source
ESI (Electro spray ionization)
Polarity
Positive
Nebulizer gas
N2
Nebulizer pressure
36 psi
Gas flow
11 L/min
Ion spray voltage
3500 V
Source temp.
300 ℃
Resolution
Q1(unit) Q3(unit)
Scan mode
MRM (Multiple reation monitoring)
MRM condition Retention Time(min) 10.7
Precursor Compound ion
D3
(m/z)
Dwell Fragmentor Collision (ms)
(V)
Energy(V)
385
107 Quantitative 200
115
24
385
159 Qualitative
200
115
22
385
259 Qualitative
200
115
10
(m/z) Vitamin
Product ion
379 380
27
381
Table 2
382
Recovery and monitoring tests for vitamin D3 in certified reference material (SRM 1849a) and
383
infant formulas using no-heating saponification method with liquid chromatography-tandem
384
mass spectrometry analysis. Tested Valuea
Samples
(µg/kg)
SRM 1849ab
RSD (%)
119.50 ± 3.26
2.73
Spikedc
150.44 ± 3.64
2.72
Blank
77.95 ± 3.95
5.07
Spikedc
119.01± 1.78
2.68
Blank
61.36 ± 1.10
1.79
Spikedc
212.86 ± 3.63
2.93
Infant formula sample 1
Recovery (%)
107.65
93.11
Infant formula sample 2
93.95
Infant formula sample 3
102.86 Blank
104.93 ± 0.95
0.91
Spikedc
255.43 ± 4.76
2.66
Infant formula sample 4
110.65 Blank
121.02 ± 0.72
385 386
a
The values are mean ±S.D of 3 replications.
b
The certificated value of SRM 1849a was 111 ± 17 µg/kg.
387
c
The spiked levels of infant formula was 100 µg/kg.
388 389
28
0.59
390 391
Highlights
392 393
* No-heating saponification and dSPE clean-up was applied to extract trace vitamin D3 from
394
milk-based infant formula.
395
*Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for accurate
396
quantitative analysis.
397
*The result of recovery test using certified reference material showed 93.11-110.65%.
398
29
