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Estimation of Beta-Sitosterol in Milk Fat (Ghee) by Agilent 8890 GC and 5977B MS

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Estimation of Beta-Sitosterol in Milk Fat (Ghee) by Agilent 8890 GC and 5977B MS Application Note Food Testing Estimation of β-Sitosterol in Milk Fat (Ghee) Samples Agilent 8890/5977B Single Quadrupole GC/MS System Authors Abstract Praveen Arya, Vivek Dhyani, This application note demonstrates the use of the Agilent 8890 GC and the and Dr. Samir Vyas Agilent 5977B GC/MS single quadrupole mass spectrometer in the detection Agilent Technologies, Inc. and quantification of β-sitosterol in milk fat samples to check for vegetable oil adulteration. The method provides the highest confidence for routine analysis of milk fat samples in the food industry, whether it is used in manufacturing, processing, commercial testing, or academia. Sample preparation for this method involved saponification, followed by extraction of unsaponifiable matter by liquid-liquid extraction (LLE) and derivatization of sitosterol to its trimethylsilyl derivative. Introduction Experimental conditions Ghee sample preparation 1. Take 200 mg of fat sample in a Ghee (milk fat), also known as clarified Chemicals required 15 mL screw cap tube. butter, is commonly used in cuisine 20% ascorbic acid (AR grade) (2 g of of the Indian subcontinent, traditional ascorbic acid dissolved in 10 mL water, 2. Add saponification mixture (200 µL or medicines, and religious rituals. Ghee is potassium hydroxide (10 M, 56 g of KOH 20% ascorbic acid and 500 µL of prepared by skimming the milk solids 10 M KOH solution). dissolved in 100 mL of water)(AR grade), out of melted butter. Increasing demand water (Millipore, Milli-Q) n-hexane (HPLC for ghee in India has resulted in certain grade) β-sitosterol reference standard, 3. Vortex for 5 minutes. malpractices such as adulteration of 1 BSTFA reagent (N,O-bis(trimethylsilyl) ghee with vegetable oils. 2 trifluro), pyridine (GC grade). 4. Place in a water bath (45 °C/30 min), cooled. Plant sterols can be used as marker All working solutions of β-sitosterol were compounds for identification of prepared in n-hexane. 100 µL of standard 5. Add H O:hexane mixture (1:5) vegetable oil adulteration in ghee. One 2 was placed into a vial, before 50 µL of and vortex for 10 minutes. of the common representatives of plant pyridine was added. The vials were kept sterol is β-sitosterol. at 80 °C for 40 minutes. The final extract 6. Remove the upper 1 mL of the hexane layer was taken in vial inserts and injected into into another tube (centrifuge if necessary). the GC/MS. 7. Add 0.5 mL H O and vortex again. Instrument 2 Agilent 8890 GC system with S/SL inlet and Agilent 5977B GC/MS single 8. Take 100 µL of the upper extract in a vial and add 50 µL of BSTFA and 50 µL of pyridine. quadrupole 9. Incubate at 80 °C for 40 minutes. Figure 1. The structure of β-sitosterol. 10. Transfer the extract in a vial insert This application note demonstrates and inject into the GC/MS. detection of vegetable oil adulteration Figure 2. Liquid-liquid extraction, sample in ghee. The principle of detection of preparation. adulteration is based on the presence of β-sitosterol as a marker in the Table 1. GC method. unsaponifiable matter of pure ghee and GC Conditions adulterated ghee samples.3 Column Agilent J&W HP-5ms Ultra Inert, 30 m × 0.25 mm, 0.25 μm (p/n 19091s-433UI) The method used for detection of the Agilent split/splitless inlet presence of any vegetable oil is based Inlet 5190-2293, splitless liner Injection volume: 1 µL on gas chromatography coupled to Split Mode And Ratio Split ratio 5:1 mass spectrometry. Inlet Temperature 280 °C Oven 80 °C for 1 minute, at 15 °C/min to 290 °C, hold 30 minutes Carrier Gas 99.9995% helium at 1.0 mL/ min, constant flow mode Table 2. MS method. MSD Conditions Quadrupole Temperature 150 °C Ion Source Temperature EI 230 °C Transfer Line Temperature 290 °C Acquisition Type SIM mode; ions – 396, 486, 357, 381, 129 EMV Mode Delta EMV: 0 Dwell Time for Each Mass 50 2 396.0, 357.0, 381.0 Results and discussion ×102 ×102 With the method described, instrument 2.4 ) 1.0 LOQ was determined at 200 ppb 2.2 0.8 for the reference standard. Figure 3 2.0 highlights the quantifier and qualifier 1.8 0.6 EIC at LOQ level. Figure 4 describes the Counts 1.6 0.4 signal-to-noise for 200 and 500 ppb level 1.4 0.2 standards. Figure 4 also showcases the 1.2 Relative abundance (% specificity of the method by showing a 1.0 0 blank injection. 0.8 -0.2 22.0 22.5 23.0 22.0 22.5 23.0 Acquisition time (min) Acquisition time (min) Figure 3. Qualifier and quantifier peaks of sitosterol at LOQ (200 ppb). ×102 Noise (peak-to-peak) = 4.5605, SNR (22.410 min) = 35.7 4 Counts 2 ×102 Noise (peak-to-peak) = 4.8595, SNR (22.410 min) = 76.0 4 Counts 2 ×102 Noise (peak-to-peak) = 9.2964 4 Counts 2 21 22 23 24 25 26 27 28 29 Acquisition time (min) Figure 4. Sensitivity of β-sitosterol at 200 ppb (LOQ), 500 ppb, and blank. 3 Calibration and linearity ×103 A linearity plot was generated 4.5 for response (peak area) across 4.0 concentration levels from 200 ppb to 5 ppm (figure 6). Calibration was 3.5 performed at five levels, 200 ppb, 3.0 500 ppb, 1 ppm, 2 ppm, and 5 ppm. Linearity with R2 >0.999 was observed. 2.5 The calibration table with one quantifier Counts 2.0 ion and two qualifier ions is shown 1.5 in Figure 7, in accordance with the regulations. 1.0 0.5 0 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0 Acquisition time (min) Figure 5. Overlay of various concentrations of β-sitosterol. ×104 β-Sistosterol 2.0 y = 4207.166813x – 116.913527 1.8 R2 = 0.9993 1.6 1.4 1.2 1.0 Response 0.8 0.6 0.4 0.2 0 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Concentration (ppm) Figure 6. Calibration plot for β-sitosterol standards (concentration (ppm) versus response (peak area)). Figure 7. Calibration table for β-sitosterol from 200 ppb to 5 ppm and repeatability at 1 ppm. 4 Repeatability Table 3. Percentage RSD (CV) for peak area at 1 ppm β-sitosterol. A repeatable response was obtained by Area Inj-1 Area Inj-2 Area Inj-3 Area Inj-4 Area Inj-5 Area Inj-6 %RSD injecting 1 ppm β-sitosterol standard. 4794 4894 4971 5082 5006 5132 2.48 As shown in Table 3, the % RSD data of β-sitosterol are calculated from peak areas of six replicate injections at 1 ppm concentration. Quantitation in ghee samples The suggested method was extended Figure 8. Quantitation in ghee samples. to a ghee sample. The ghee sample was purchased from a market for the analysis and recovery study. Table 4. Recovery in ghee samples. As shown in Figure 8, 2.24 ppm Spiked Amount Observed Amount Final Amount Recovery β-sitosterol was found in the ghee (ppm) (ppm) (ppm) (%) sample on which the study was 5 8.52 6.28 125.6 performed. A chromatogram of ghee 25 23.1 20.86 83.4 sample is shown in Figure 9. Recovery study Spiking was done at 5 and 25 ppm respectively. Table 4 shows recovery in the spiked samples after blank subtraction. 396.0, 357.0, 381.0 ×103 ×102 ×103 0.9 ) 2.5 3.5 0.8 2.0 3.0 0.7 1.5 2.5 0.6 1.0 0.5 2.0 Counts Counts 0.4 0.5 1.5 0.3 0 1.0 0.2 Relative abundance (% -0.5 0.5 0.1 -1.0 0 22.0 22.5 23.0 22.0 22.5 23.0 150 200 250 300 350 400 450 500 Acquisition time (min) Acquisition time (min) Mass-to-charge (m/z) Figure 9. Chromatogram for ghee sample spiked at 25 ppm level for sitosterol. 5 Conclusion References An accurate and rugged method was 1. FSSAI releases Methods of developed for analysis of β-sitosterol Detecting Adulteration in Ghee, in ghee samples for the identification Food Safety Mantra, May 27, 2019. of adulteration by vegetable oils. The https://www.foodsafetymantra. saponification/LLE/derivatization based com/2019/05/fssai-releases- sample preparation method used easy methods-of-detecting-adulteration- and less time-consuming steps. The in-ghee. lowest calibration level was 200 ppb for 2. Jyotika. Profiling of milk fat of the standard. Repeatable results were different species of milch animals. found for six replicates of standard. Publisher: NDRI, Karnal. Citation: Satisfactory recovery was found at 5 P-1452, 2016. and 25 ppm spiked concentration in ghee samples. 3. Rani, A. et al. A Rapid Reversed- Phase Thin Layer Chromatographic Protocol for Detection of Adulteration in Ghee (Clarified Milk Fat) with Vegetable Oils. Journal of Food Science and Technology 2015, 52(4), 2434–2439. www.agilent.com/chem DE4602546296 This information is subject to change without notice. © Agilent Technologies, Inc. 2020 Printed in the USA, November 10, 2020 5994-2725EN.
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