Research articlesSalting-out assisted liquid–liquid extraction coupled with high-performance liquid chromatography for the determination of vitamin D3 in milk samples
Abstract
In this study, salting-out assisted liquid–liquid extraction (SALLE) as a simple and efficient extraction technique followed by high-performance liquid chromatography (HPLC) was employed for the determination of vitamin D3 in milk samples. The sample treatment is based on the use of water-miscible acetonitrile as the extractant and acetonitrile phase separation under high-salt conditions. Under the optimum conditions, acetonitrile and ammonium sulfate were used as the extraction solvent and salting-out agent, respectively. The vitamin D3 extract was separated using Hypersil ODS (250x i.d 4.6 mm, 5 µm) HPLC column that was coupled with diode array detector. Vitamin D2 was used as internal standard (IS) to offset any variations in chromatographic conditions. The vitamin D3 and the IS were eluted in 18 min. Good linearity (r2 > 0.99) was obtained within the range of 25–600 ng g−1 with the limit of detection of 15 ng g−1 and limit of quantification of 25 ng g−1. The validated method was applied for the determination of vitamin D3 in milk samples. The recoveries for spiked samples were from 94.4 to 113.5%.
1. Introduction
Cholecalciferol, commonly known as vitamin D3, is a fat-soluble vitamin, which is of great nutritional interest, that supports metabolism processes and improves the efficiency of proteins and enzymes [1]. It plays important roles within the body as it regulates blood calcium and phosphorus levels by promoting the absorption in intestines and reabsorption of calcium in the kidney that is key to the mineralization of the bones [2,3]. Vitamin D deficiency is recognized as one of the most common mild chronic medical conditions in the world. It can lead to soft, thin and brittle bones, a disease known as rickets in children [4,5].
Vitamin D3 is naturally found in trace amounts in some foods such as some fatty fishes (mackerel, salmon and sardines), fish liver oils and eggs from hens that have been fed vitamin D3. Among dairy products, processed milk products and infant formulas are fortified with vitamin D3. However, its content is low, ranging from 4 to 40 IU (0.1–1.0 µg l−1) and which corresponds mainly to vitamin D3 and 25-hydroxy-vitamin D3 [6]. Vitamin D3 is also sensitive to heat and light and is easily oxidized [7].
Sample preparation process plays an important role in enhancing sensitivity and reducing matrix interference. The most common preparation technique is saponification [8,9], liquid–liquid extraction (LLE) [10], solid-phase extraction (SPE) [11–14] and derivatization [15]. Saponification is commonly the first step before LLE and SPE and is used to remove neutral lipids, especially triglycerides. This method has been used for the determination of vitamin D in various types of food samples such as infant products [12,16], milk, cream and butter [17], human foods, pet foods and supplements [18], fish oil [19], beef and poultry [20], fortified orange juice [21], dietary supplements and vitamin premixes [22], bovine milk [11], vegetables [23], meat [24] and human serum [6]. Saponification using methanolic or ethanolic KOH at elevated temperatures (60–100°C) for 20–45 min was able to suppress lipids and proteins interferences [25]. A disadvantage of the heat treatment is the possibility of isomerization of vitamin D3 and this causes difficulty in liquid chromatography (LC) analysis.
LLE and SPE involve high consumption of solvents during the multi-step extraction and the long-time sample preparation. Research in vitamin D3 chemistry needs to be supported by the development of rapid and sensitive modern analytical methods and efficient sample clean-up procedures. Quantification of vitamin D3 in dairy products has been considered one of the most difficult goals because of its low content in complex matrix [25].
Salting-out assisted liquid–liquid extraction (SALLE) is another interesting approach for extraction of vitamin D3. Salting-out is a process of addition of electrolytes to an aqueous phase to increase the distribution of a solute. SALLE can be viewed as modification to the conventional LLE procedure so that the extract is compatible for LC analysis. In the SALLE procedure, water-miscible organic solvents such as acetonitrile (ACN) and salts such as sodium chloride or ammonium sulfate are added. After gentle mixing and centrifugation, the organic solvent is ‘salted-out' and formed a separate phase on the top of the aqueous phase. The extract can be conveniently sampled and injected directly into LC for analysis.
When this sample pretreatment method was applied for the determination of aflatoxins in milk samples, the analytes were not only extracted but proteins were precipitated, separated the fats from the water-soluble components (lactose and minerals) [26]. It is noted that most applications reported with SALLE method are focused on the extraction of polar compounds [27,28].
It is clear from the above discussions that alternative sample pretreatments that are simpler but maintaining the integrity of the vitamins D2 and D3 are required. Efforts should be directed towards eliminating the saponification step and performing at room temperature to prevent isomerism and decomposition. Towards this end, of the plethora of microextraction techniques, the SALLE seemed to be the best candidate. Thus, this paper is dedicated to the development of a SALLE pretreatment method for the determination of vitamin D3 in milk.
2. Experimental
2.1. Chemicals and reagents
The analytical standard of vitamin D3, high-performance liquid chromatography (HPLC)-grade methanol and acetonitrile were purchased from Merck (Darmstadt, Germany). Vitamin D2 and vitamin C (L-ascorbic acid) were obtained from Sigma-Aldrich (St Louis, MO, USA). Analytical grade ethanol (denatured) and ammonium sulfate were purchased from QReC (Auckland, New Zealand). Formic acid (99%) was supplied by Univar (Auburn, Australia). Distilled water (resistivity, 18.2 mΩ cm−1) was produced by Milipore water purification system (Molsheim, France) and was used throughout for the preparation of solutions. Vitamin D3 and D2 stock solutions (1000 ng g−1) in methanol were used to prepare standard solutions.
2.2. Instrumentation
HPLC Perkin Elmer Series 200 (Waltham, MA, USA) with diode-array detector (DAD) were used. Separation was performed on Hypersil ODS (250x i.d 4.6 mm, 5 µm) from Thermo Fisher Scientific (Waltham, MA, USA). Methanol : water (98 : 2 (v/v) %) as mobile phase at flow rate 0.8 ml min−1 were used. The acquisition of data was done at 265 nm for both vitamins (D3 and D2). The injection volume used for the determination was 100 µl and both vitamins were eluted within 18 min. The process was controlled by TotalChrom™ software.
2.3. Vitamin D3 stability tests
Preliminary experiments were conducted to test the stability of vitamin D3 after exposure to external factors such as light, elevated temperature and hydrogen peroxide (oxidizing agent). The standard vitamin D3 was stored in different storage conditions: 25°C or 40°C exposed to light, and with addition of hydrogen peroxide (2–10 v/v %). Immediately after collection, the standards from each condition were analysed using HPLC. The stability of vitamin D3 under these conditions was determined by calculating the percentage change in concentration relative to fresh sample.
2.4. SALLE procedure
The milk samples were obtained from local stores in Gelugor, Penang, Malaysia. Milk samples (5 g) were weighed and dissolved in distilled water (10 ml) at room temperature. The mixtures were sonicated for 30 min in order to ensure that the milk samples were completely dissolved. In order to prevent analyte loss due to the light and temperature effects, the mixtures were covered with aluminium foils and sonicated at room temperature. After 30 min sonication, ACN (3 ml) was added followed by the addition of vitamin C in ethanol [16] into the mixtures (150 µl). Next, the mixtures were sonicated (60 min) and mixed for 10 min. About 7 g of ammonium sulfate ((NH4)2SO4) and formic acid (50 µl) were added into the mixtures. The mixtures were then mixed well until the salt was completely dissolved before centrifuging (8500 r.p.m.) for 30 min. The upper layer (about 1 ml) was transferred into small vials and evaporated using N2 stream. The residues were reconstituted using a mobile phase (150 µl) before injected into the HPLC system.
2.5. Method validation
The method was validated for linearity, detection and quantification limits, repeatability and recoveries. The linearity of the method was assessed by plotting a calibration curve over 25–600 ng g−1 of standard vitamin D3. The detection and quantification limits were determined from the slope of calibration curve. For a linear calibration curve, it can be expressed by equation, y = a + bx. In this model, the sensitivity, b and the limit of detection (LOD) can be determined as
The determination of recovery was performed by spiking three different concentrations of standard vitamin D3 in milk samples. The recovery was calculated based on the measured vitamin D3 concentration data as shown below:
3. Result and discussion
3.1. Preliminary studies of vitamin D3 stability
Vitamin D3 is known to degrade under various conditions; thereby its analysis is considered challenging. Preliminary tests were conducted to test the stability of vitamin D3 after exposure to the light, elevated temperature and oxidizing agent under the present experimental conditions. These tests were necessary to provide information on the handling of reagents prior to the SALLE procedure. About 65% of vitamin D3 was degraded after being exposed to these conditions, in agreement with earlier studies [29]. Thus, it was decided that an antioxidant (ascorbic acid) would be used during the SALLE procedure.
3.2. Effect of type of extraction solvent
The type of extraction solvent is an important parameter in SALLE procedure. The ideal extraction solvent should be miscible in water, polar and able to induce phase separation upon addition of salt [27]. In this study, tetrahydrofuran (THF) and acetonitrile (ACN) were tested as extraction solvents. ACN gave better results compared to THF for the extraction of vitamin D3 as shown in figure 1a. ACN is a highly recommended solvent because it has high polarity and is miscible in water. Upon the addition of salts, ACN is able to precipitate proteins and caused partition of the analyte into organic solvent. Besides, ACN is a less harmful organic solvent compared to THF.
Figure 1. Optimization of salting out liquid–liquid extraction parameters (a) type of extraction solvents; (b) volume of ACN; (c) type of salt; (d) amount of salt. Extraction condition: sample volume, 10 ml; time of sonication, 60 min.
3.3. Effect of volume of extraction solvent
The mass transfer of the analytes is expected to increase as the volume of ACN is increased due to the enhancement of partitioning of analytes between the two phases. Different volumes of the extraction solvent (2–4 ml) were investigated (figure 1b). It was found that the peak areas gradually increased as the volume of ACN increased up to 3 ml and decreased afterwards due to dilution factor effect. This is because at a lower volume (2 ml), the separated organic phase was not obvious, and the collection of the organic phase was difficult. Thus, 3.0 ml of ACN was selected for the extraction of vitamin D3 from milk samples.
3.4. Effect of addition of salts
The type of salting-out reagent is an important consideration in the SALLE procedure. Different type and amount of salts affect the degrees of phase separation. In this study, the effects of sodium chloride and ammonium sulfate were evaluated. The results demonstrated that ammonium sulfate provided better phase separation and higher peak areas compared to sodium chloride (figure 1c). Ammonium sulfate ((NH4)2SO4) is often used as salting-out reagent because of its high solubility, allowing for solutions to be prepared at high ionic strength, low price and its easy availability [30]. Thus, (NH4)2SO4 was selected for the subsequent study. The effect of different amounts of (NH4)2SO4 (3.0–9.0 g) on the extraction efficiency was also investigated. The optimum amount of salt was 7.0 g (figure 1d). The peak area of the extracted vitamin D3 increased with increasing amount of salt up to 7.0 g and the area decreased thereafter.
3.5. Effect of sonication time
Sonication treatment has two main purposes, i.e. to improve the extraction yields and to accelerate the phase separation. After the addition of ACN into the sample solution, the effect of sonication was examined. The extraction yields of vitamin D3 obtained at different times of sonication (30 and 60 min) showed an increase in peak area as a function of time. Sonication treatment for 60 min provides good extraction of vitamin D3 from the aqueous phase into the ACN phase. Sonication treatment more than that should be avoided, because the temperature of the sample solution started to increase, which could cause analyte loss due to excessive heating, as the stability of vitamin D3 was affected by temperature at prolonged time. For subsequent analysis, 60 min of sonication was chosen.
3.6. Adopted extraction conditions
The adopted extraction conditions for 10 ml sample were performed with 3 ml ACN as the extraction solvent and 7.0 g of (NH4)2SO4 salt as the salting-out reagent with 60 min of sonication.
3.7. Method validation
Under the above optimum experimental conditions, the proposed SALLE method was validated in terms of linearity, LOD, limits of quantification (LOQ), repeatability and recovery. The calibration curve was obtained by linear regression of peak area ratio of D3 over internal standard (IS), versus concentration of vitamin D3 (25–600 ng g−1). Typical plot is described by
Recovery was evaluated by spiking the milk samples with standard vitamin D3 at different concentrations (table 1). The recoveries were acceptable (94.4–113.5%). The precision of the analytical procedure was determined using six replicates samples. The repeatability obtained was good (below 15%). It was found that the precision (% r.s.d.) for three concentration levels (50, 200 and 600 ng g−1) was less than 11.5%.
| concentration spiked (ng g−1) | recovery % (average ± s.d. %) | precision (% r.s.d.) |
|---|---|---|
| 50 | 94.40 ± 10.8 | 11.4 |
| 200 | 113.5 ± 1.78 | 1.56 |
| 600 | 106.6 ± 4.34 | 4.07 |
3.8. Analysis of milk samples
In order to evaluate the applicability of the proposed method, different milk samples were analysed and results were compared with the amount claimed by manufacturers (table 2). All analysed samples contained vitamin D3 concentrations that were well as reported by the manufacturers. The average tested values for milk samples were in range 18.1–69.0 ng g−1. The recoveries obtained ranged from 64.5 to 102.5%, and the relative standard deviation (r.s.d.) values ranged from 2.8 to 17.9%.
| sample | proposed method (ng g−1) (average ± s.d.) | recovery (%) | r.s.d. (%) |
|---|---|---|---|
| milk powder 1 | 43.4 ± 0.03 | 102.5 | 6.5 |
| milk powder 2 | 69.0 ± 0.001 | 73.9 | 17.9 |
| milk powder 3 | 48.0 ± 0.005 | 95.4 | 9.9 |
| milk powder 4 | 48.1 ± 0.004 | 96.2 | 8.5 |
| liquid milk 1 | 38.8 ± 0.004 | 64.5 | 9.9 |
| liquid milk 2 | 21.6 ± 0.001 | 86.0 | 4.1 |
| liquid milk 3 | 20.2 ± 0.002 | 80.4 | 8.0 |
| liquid milk 4 | 18.1 ± 0.001 | 69.6 | 2.8 |
A student paired t-test (at 95% confidence level) shows that there is any significant difference in the mean values of the manufacturer's claimed values and that of the proposed method (t stat, 0.08; t critical, 2.2).
3.9. Comparison to previously reported methods
Table 3 summarizes the important characteristics of proposed method for the determination of vitamin D3 in milk samples compared to some previously reported methods. It is readily apparent that the developed method using SALLE has many advantageous features especially in terms of solvent consumption, sample preparation time and sensitivity. This proposed method is simple, since there is no saponification method involved, unlike some previously reported methods. Some of the previously reported methods use SPE, but this requires multi-steps (equilibration, washing, loading and elution) with single-use SPE cartridges, which makes the experiment longer.
| samples | extraction technique | extraction time (min) | technique | LOQ (ng g−1) | recovery (%) | ref |
|---|---|---|---|---|---|---|
| milk product (solid, liquid) | SALLE | 60 min (without saponification) | HPLC-DAD | 25 | 94.4–113.5 | this work |
| milk product (solid, liquid) | LLE | 70 min saponification + extraction time | LC-UV | 100 | 93.0–102.0 | [16] |
| infant formula | DLLME | — | LC-MS | 1 | 88.0–103.0 | [23] |
| infant formula | LLE dSPE | 10 min saponification + extraction time | LC-MSMS | 1 | 93.1–110.6 | [12] |
| infant formula | LLE | overnight saponification + 10 min extraction time | HPLC-MSMS | 5 | 89.5–115.0 | [18] |
| milk product (solid) | LLE | overnight saponification + 20 min extraction time | HPLC-DAD | 20 | 73.0–115.0 | [31] |
| milk product (solid) | packed fibre SPE | — | HPLC-DAD | 70 | 91.7–104.5 | [22] |
| cod liver fish oil supplement | SPE | — | LC-MS | 0.1 | 88.8–99.3 | [19] |
4. Conclusion
Significant advancement is sample pretreatment method based on SALLE for the HPLC determination of vitamin D3 in milk is demonstrated. This method is simple, fast and consumes less amount of solvent compared to previous sample pretreatment methods which require saponification and elevated temperatures. The extracts are readily injected for the HPLC analysis, unlike previous methods that require long and gentle evaporation to remove extracting solvents. It is indeed very interesting if the SALLE technique can be adopted, with minor modifications, to be applied to the analysis of other physiologically important metabolites of vitamin D such as 25-OH-D. If this is possible, it will be a major breakthrough in the analysis of vitamin D metabolites.
Data accessibility
Data are available from the Dryad Digital Repository: https://doi.org/10.5061/dryad.cq3bp7m [32].
Authors' contributions
K.Y.C., M.M.C., A.A., B.S. and M.M. participated in the design of the study; N.H.S., K.Y.C. and M.M.C. carried out most of the laboratory work, accomplished the data analysis and drafted the manuscript; M.M., A.A. and B.S. conceived the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
Competing interests
The authors declare that they have no competing interests.
Funding
This work was supported by funding from Universiti Sains Malaysia Research Grants (Short Term: 304.PKIMIA.6313334; Bridging: 304.PKIMIA.6316492).
Acknowledgements
The authors thank the editors and anonymous reviewers for their helpful suggestions for this manuscript.
Footnotes
References
- 1.
Płonka J, Toczek A, Tomczyk V . 2012Multivitamin analysis of fruits, fruit–vegetable juices, and diet supplements. Food Anal. Methods 5, 1167-1176. (doi:10.1007/s12161-011-9349-3) Crossref, ISI, Google Scholar - 2.
Reid IR, Bolland MJ, Grey A . 2014Effects of vitamin D supplements on bone mineral density: a systematic review and meta-analysis. Lancet 383, 146-155. (doi:10.1016/S0140-6736(13)61647-5) Crossref, PubMed, ISI, Google Scholar - 3.
Raml R, Ratzer M, Obermayer-Pietsch B, Mautner A, Pieber TR, Sinner FM, Magnes C . 2015Quantifying vitamin D and its metabolites by LC/orbitrap MS. Anal. Methods 7, 8961-8966. (doi:10.1039/C5AY01583A) Crossref, ISI, Google Scholar - 4.
Holick MF . 2007Vitamin D deficiency. N. Engl. J. Med. 357, 266-281. (doi:10.1056/NEJMra070553) Crossref, PubMed, ISI, Google Scholar - 5.
Jenkinson C, Bradbury J, Taylor A, Adams JS, He S, Viant MR, Hewison M . 2017Automated development of an LC-MS/MS method for measuring multiple vitamin D metabolites using MUSCLE software. Anal. Methods 9, 2723-2731. (doi:10.1039/C7AY00550D) Crossref, ISI, Google Scholar - 6.
Fang H, Yu S, Cheng Q, Cheng X, Han J, Qin X, Xia L, Jiang X, Qiu L . 2016Determination of 1,25-dihydroxyvitamin D2 and 1,25-dihydroxyvitamin D3 in human serum using liquid chromatography with tandem mass spectrometry. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 1027, 19-26. (doi:10.1016/j.jchromb.2016.04.034) Crossref, PubMed, ISI, Google Scholar - 7.
Mahmoodani F, Perera CO, Fedrizzi B, Abernethy G, Chen H . 2017Degradation studies of cholecalciferol (vitamin D3) using HPLC-DAD, UHPLC-MS/MS and chemical derivatization. Food Chem. 219, 373-381. (doi:10.1016/j.foodchem.2016.09.146) Crossref, PubMed, ISI, Google Scholar - 8.
Dalla Nora FM, Oliveira AS, Lucas BN, Ferreira DDF, Duarte FA, Mello RO, Cichoski AJ, Barin JS . 2018Miniaturized, high-throughput and green determination of the saponification value of edible oils using thermal infrared enthalpimetry. Anal. Methods 10, 3770-3776. (doi:10.1039/C8AY00597D) Crossref, ISI, Google Scholar - 9.
Xie Q, Sun D, Cao J, Jia L, Hou B, Li D . 2016Determination of α-tocopherol in cereal grains by use of saponification coupled with ionic-liquid-based dispersive liquid-liquid microextraction before high-performance liquid chromatography-mass spectrometry. Anal. Methods 8, 5283-5287. (doi:10.1039/C6AY01112H) Crossref, ISI, Google Scholar - 10.
Han Y, Jia X, Duan T, Chen H . 2011Combination of saponification with in-tube liquid-liquid extraction and dispersive liquid-liquid microextraction for determination of polybrominated diphenyl ethers in whole milk by gas chromatography-mass spectrometry. Anal. Methods 3, 842-848. (doi:10.1039/c0ay00742k) Crossref, ISI, Google Scholar - 11.
Trenerry VC, Plozza T, Caridi D, Murphy S . 2011The determination of vitamin D3 in bovine milk by liquid chromatography mass spectrometry. Food Chem. 125, 1314-1319. (doi:10.1016/j.foodchem.2010.09.097) Crossref, ISI, Google Scholar - 12.
Kwak B-M, Jeong I-S, Lee M-S, Ahn J-H, Park J-S . 2014Rapid determination of vitamin D3 in milk-based infant formulas by liquid chromatography-tandem mass spectrometry. Food Chem. 165, 569-574. (doi:10.1016/j.foodchem.2014.05.137) Crossref, PubMed, ISI, Google Scholar - 13.
Abro K, Memon N, Bhanger MI, Abro S, Perveen S, Lagharì AH . 2014Determination of vitamins E, D3, and K1 in plasma by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry utilizing a monolithic column. Anal. Lett. 47, 14-24. (doi:10.1080/00032719.2013.831424) Crossref, ISI, Google Scholar - 14.
Xiao X, Zhang M-M, Wang Z-Q . 2018Determination of β-blockers in bovine and porcine tissues and bovine milk by high-performance liquid chromatography–tandem mass spectrometry. Anal. Lett. 52, 1-13. (doi:10.1080/00032719.2018.1470638) ISI, Google Scholar - 15.
Bigdelifam D, Hashemi M, Zohrabi P, Sadeghpour M, Radaee E . 2017Sensitive magnetic dispersive solid-phase extraction using hydrophobic magnetic nanoparticles and GC-MS analysis for the determination of sterol composition in milk samples for the detection of palm oil. Anal. Methods 9, 2211-2219. (doi:10.1039/C7AY00459A) Crossref, ISI, Google Scholar - 16.
Staffas A, Nyman A . 2003Determination of cholecalciferol (Vitamin D3) in selected foods by liquid chromatography: NMKL collaborative study. J. AOAC Int. 86, 400-406. Crossref, PubMed, ISI, Google Scholar - 17.
Jakobsen J, Saxholt E . 2009Vitamin D metabolites in bovine milk and butter. J. Food Compos. Anal. 22, 472-478. (doi:10.1016/j.jfca.2009.01.010) Crossref, ISI, Google Scholar - 18.
Huang M, LaLuzerne P, Winters D, Sullivan D . 2009Measurement of vitamin D in foods and nutritional supplements by liquid chromatography/tandem mass spectrometry. J. AOAC Int. 92, 1327-1335. Crossref, PubMed, ISI, Google Scholar - 19.
Bartolucci G, Giocaliere E, Boscaro F, Vannacci A, Gallo E, Pieraccini G, Moneti G . 2011Vitamin D3 quantification in a cod liver oil-based supplement. J. Pharm. Biomed. Anal. 55, 64-70. (doi:10.1016/j.jpba.2011.01.007) Crossref, PubMed, ISI, Google Scholar - 20.
Bilodeau L, Dufresne G, Deeks J, Clément G, Bertrand J, Turcotte S, Robichaud A, Beraldin F, Fouquet A . 2011Determination of vitamin D3 and 25-hydroxyvitamin D3 in foodstuffs by HPLC UV-DAD and LC-MS/MS. J. Food Compos. Anal. 24, 441-448. (doi:10.1016/j.jfca.2010.08.002) Crossref, ISI, Google Scholar - 21.
Byrdwell WC, Exler J, Gebhardt SE, Harnly JM, Holden JM, Horst RL, Patterson KY, Phillips KM, Wolf WR . 2011Liquid chromatography with ultraviolet and dual parallel mass spectrometric detection for analysis of vitamin D in retail fortified orange juice. J. Food Compos. Anal. 24, 299-306. (doi:10.1016/j.jfca.2010.09.020) Crossref, ISI, Google Scholar - 22.
Chen L, Liu Z, Kang X, Zhou X, Zheng S, Gu Z . 2011Determination of fat-soluble vitamins in food and pharmaceutical supplements using packed-fiber solid phase extraction (PFSPE) for sample preconcentration/ clean-up. Procedia Environ. Sci. 8, 588-595. (doi:10.1016/j.proenv.2011.10.091) Crossref, Google Scholar - 23.
Vinas P, Bravo-Bravo M, Lopez-Garcia I, Hernandez-Cordoba M . 2013Dispersive liquid-liquid microextraction for the determination of vitamins D and K in foods by liquid chromatography with diode-array and atmospheric pressure chemical ionization-mass spectrometry detection. Talanta 115, 806-813. (doi:10.1016/j.talanta.2013.06.050) Crossref, PubMed, ISI, Google Scholar - 24.
Strobel N, Buddhadasa S, Adorno P, Stockham K, Greenfield H . 2013Vitamin D and 25-hydroxyvitamin D determination in meats by LC-IT-MS. Food Chem. 138, 1042-1047. (doi:10.1016/j.foodchem.2012.08.041) Crossref, PubMed, ISI, Google Scholar - 25.
Kasalová E, Aufartová J, Krčmová LK, Solichová D, Solich P . 2015Recent trends in the analysis of vitamin D and its metabolites in milk: a review. Food Chem. 171, 177-190. (doi:10.1016/j.foodchem.2014.08.102) Crossref, PubMed, ISI, Google Scholar - 26.
Magiera S, Kwietniowska E . 2016Fast, simple and efficient salting-out assisted liquid-liquid extraction of naringenin from fruit juice samples prior to their enantioselective determination by liquid chromatography. Food Chem. 211, 227-234. (doi:10.1016/j.foodchem.2016.05.045) Crossref, PubMed, ISI, Google Scholar - 27.
Tang YQ, Weng N . 2013Salting-out assisted liquid–liquid extraction for bioanalysis. Bioanalysis 5, 1583-1598. (doi:10.4155/bio.13.117) Crossref, PubMed, ISI, Google Scholar - 28.
Campone L, Piccinelli AL, Celano R, Pagano I, Russo M, Rastrelli L . 2016Rapid and automated analysis of aflatoxin M1 in milk and dairy products by online solid phase extraction coupled to ultra-high-pressure-liquid-chromatography tandem mass spectrometry. J. Chromatogr. A 1428, 212-219. (doi:10.1016/j.chroma.2015.10.094) Crossref, PubMed, ISI, Google Scholar - 29.
Kamankesh M, Shahdoostkhany M, Mohammadi A, Mollahosseini A . 2018Fast and sensitive low density solvent-based dispersive liquid-liquid microextraction method combined with high-performance liquid chromatography for determining cholecalciferol (vitamin D3) in milk and yogurt drink samples. Anal. Methods 10, 975-982. (doi:10.1039/C7AY02915B) Crossref, ISI, Google Scholar - 30.
Duong-Ly KC, Gabelli SB . 2014Laboratory methods in enzymology: protein part C, pp. 85-94. Cambridge, MA: Academic Press. Google Scholar - 31.
Kienen V, Costa WF, Visentainer JV, Souza NE, Oliveira CC . 2008Development of a green chromatographic method for determination of fat-soluble vitamins in food and pharmaceutical supplement. Talanta 75, 141-146. (doi:10.1016/j.talanta.2007.10.043) Crossref, PubMed, ISI, Google Scholar - 32.
Sazali N, Alshishani A, Saad B, Chew K, Chong M, Miskam M . 2019Data from: Salting-out assisted liquid liquid extraction coupled with high performance liquid chromatography for the determination of vitamin D3 in milk samples . Dryad Digital Repository. (doi:10.5061/dryad.cq3bp7m) Google Scholar
