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Determination of 17 Hormone Residues in Milk by Ultra-High-Performance Liquid Chromatography and Triple Quadrupole Mass Spectrom

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Determination of 17 Hormone Residues in Milk by Ultra-High-Performance Liquid Chromatography and Triple Quadrupole Mass Spectrom No. LCMSMS-065E Liquid Chromatography Mass Spectrometry Determination of 17 Hormone Residues in Milk by Ultra-High-Performance Liquid Chromatography and Triple Quadrupole No. LCMSMS-65E Mass Spectrometry This application news presents a method for the determination of 17 hormone residues in milk using Shimadzu Ultra-High-Performance Liquid Chromatograph (UHPLC) LC-30A and Triple Quadrupole Mass Spectrometer LCMS- 8040. After sample pretreatment, the compounds in the milk matrix were separated using UPLC LC-30A and analyzed via Triple Quadrupole Mass Spectrometer LCMS-8040. All 17 hormones displayed good linearity within their respective concentration range, with correlation coefficient in the range of 0.9974 and 0.9999. The RSD% of retention time and peak area of 17 hormones at the low-, mid- and high- concentrations were in the range of 0.0102-0.161% and 0.563-6.55% respectively, indicating good instrument precision. Method validation was conducted and the matrix spike recovery of milk ranged between 61.00-110.9%. The limit of quantitation was 0.14-0.975 g/kg, and it meets the requirement for detection of hormones in milk. Keywords: Hormones; Milk; Solid phase extraction; Ultra performance liquid chromatograph; Triple quadrupole mass spectrometry ■ Introduction Since 2008’s melamine-tainted milk scandal, the With reference to China’s national standard GB/T adulteration of milk powder has become a major 21981-2008 "Hormone Multi-Residue Detection food safety concern. In recent years, another case of Method for Animal-derived Food - LC-MS Method", dairy product safety is suspected to cause "infant a method utilizing solid phase extraction, ultra- sexual precocity" (also known as precocious puberty) performance liquid chromatography and triple and has become another major issue challenging the quadrupole mass spectrometry was developed for dairy industry in China. These frequent food safety the rapid and high-sensitive detection of 17 crises on milk powder quality severely decrease hormones in milk. consumer confidence in domestic milk quality as well as impede the development of milk manufacturing ■ Experimental industry. Considering the interest and motivation of 1.1. Instruments manufacturers, it is apparent that hormones are not Shimadzu Ultra-High-Performance Liquid added during milk powder manufacturing and Chromatograph (UHPLC) Nexera X2 and Triple processing. However, farmers may use antibiotics Quadrupole Mass Spectrometer LCMS-8040 system and hormones illegally on cows to increase the milk was used. The specific configuration included LC- yield for milk production. With the exception of 30AD×2 infusion pumps, DGU-20A5 Online endogenous progestogens, estrogens and Degasser, SIL-30AC Autosampler, CTO-30AC androgens, all other chemically synthesized Column Oven, CBM-20A System Controller, Triple hormones in milk belong to prohibited drugs. In Quadrupole Mass Spectrometer LCMS-8040, and 2002, the Ministry of Agriculture of China issued an LabSolutions Ver. 5.53 Chromatography announcement (no. 235) prohibiting the use of Workstation. chemically synthesized hormones such as diethylstilbestrol, medroxyprogesterone acetate, methyltestosterone, trenbolone and zeranol and stipulated that these compounds shall not be detected in any animal-derived food. At the same time, both EU directive 96/22/EC and US FDA have also banned the use of hormone drugs in animal- derived food. No. LCMSMS-065E 1.2. Analytical Conditions Liquid chromatography (LC) parameters Mass spectrometry (MS) conditions • Analyzer: Nexera X2 System • Analyzer: LCMS-8040 • Chromatographic column: Shimadzu Shim-pack XR- • Ion source: ESI, positive ion scan ODS II 2.0 mm ID x 75 mmL, 2.2µm • Ion source interface voltage: 4.5 kV • Mobile phase: A-0.1% formic acid solution, B- • Nebulizing gas: Nitrogen 3.0 L/min methanol, A/B=50%/50% (V/V) • Drying gas: Nitrogen 15 L/min • Flow rate: 0.4 mL/min • Collision gas: Argon • Elution mode: gradient elution, refer to Table 1 • DL temperature: 250°C • Injection volume: 5 μL • Heating module temperature: 400°C • Column temperature: 40°C • Scan mode: Multiple Reaction Monitoring (MRM) • Dwell time: 30 msec • Pause time: 3 msec • MRM parameters: Refer to Table 2 Table 1: Gradient Program Time(min) Module Command Value 8 Pumps PumpB Cone. 64 11 Pumps PumpB Cone. 64 12.5 Pumps PumpB Cone. 100 14.5 Pumps PumpB Cone. 100 15 Pumps PumpB Cone. 50 20 Controller Stop Table 2: MRM Parameters Q1 Pre Bias Q3 Pre Bias Compound name Precursor ion Product ion CE (V) (V) (V) 287.05 97.00* -15.0 -25.0 -20.0 Androstenedione 287.05 109.15 -15.0 -25.0 -23.0 287.10 121.05* -26.0 -25.0 -26.0 Boldenone 287.10 135.05 -26.0 -15.0 -15.0 338.25 148.15* -26.0 -30.0 -16.0 Danazol 338.25 120.00 -26.0 -35.0 -24.0 337.15 241.15* -13.0 -25.0 -18.0 Fluoxymesterone 337.15 131.00 -13.0 -35.0 -28.0 289.25 97.00* -25.0 -25.0 -20.0 Testosterone 289.25 109.05 -25.0 -25.0 -22.0 303.25 109.15* -25.0 -30.0 -21.0 Methyltestosterone 303.25 97.15 -25.0 -25.0 -19.0 301.25 121.15* -26.0 -25.0 -13.0 Methandienone 301.25 149.15 -26.0 -15.0 -16.0 275.10 109.10* -13.0 -30.0 -22.0 Nandrolone 275.10 257.20 -13.0 -15.0 -20.0 273.10 109.15* -10.0 -25.0 -23.0 Norandrostenedione 273.10 197.10 -10.0 -20.0 -15.0 271.20 253.15* -27.0 -20.0 -19.0 Trenbolone 271.20 199.05 -23.0 -25.0 -15.0 385.10 267.15* -23.0 -20.0 -20.0 Megestrol acetate 385.10 325.20 -23.0 -15.0 -25.0 345.30 123.00* -13.0 -25.0 -13.0 Medroxyprogesterone 345.30 97.20 -13.0 -25.0 -19.0 387.30 123.00* -15.0 -30.0 -13.0 Medroxyprogesterone acetate 387.30 327.25 -15.0 -10.0 -25.0 313.25 109.15* -12.0 -35.0 -24.0 Norgestrel 313.25 245.20 -12.0 -25.0 -18.0 405.05 345.20* -24.0 -15.0 -18.0 Chlormadinone acetate 405.05 309.15 -24.0 -20.0 -23.0 299.05 109.05* -19.0 -35.0 -22.0 Norethindrone 299.05 91.15 -19.0 -25.0 -11.0 315.15 97.15* -12.0 -25.0 -20.0 Progesterone 315.15 109.00 -12.0 -30.0 -21.0 * Quantifier ion No. LCMSMS-065E 1.3. Sample Preparation 1.3.1. Preparation of standard solutions subsequently diluted with water to prepare standard A mixed standard solution of 17 hormones, each working solutions at different concentration points containing 1000 µg/L, was prepared by diluting the as shown in Table 3. The standard calibration curves individual hormone standard (100 mg/L) with were established using these concentrations. ultrapure water. The mixed standard solution was Table 3: Concentrations (μg/L) of 17 Hormones used for Calibration Compound name Conc. 1 Conc.2 Conc.3 Conc.4 Conc.5 Conc.6 Conc. 7 Norandrostenedione 1 2 4 10 20 50 100 Trenbolone 1 2 4 10 20 50 100 Boldenone 1 2 4 10 20 50 100 Nandrolone 1 2 4 10 20 50 100 Androstenedione 1 2 4 10 20 50 100 Methandienone 1 2 4 10 20 50 100 Testosterone 1 2 4 10 20 50 100 Methyltestosterone 1 2 4 10 20 50 100 Medroxyprogesterone 1 2 4 10 20 50 100 Megestrol acetate 1 2 4 10 20 50 100 Medroxyprogesterone acetate 1 2 4 10 20 50 100 Progesterone 1 2 4 10 20 50 100 Norethindrone 5 10 20 50 100 250 500 Norgestrel 5 10 20 50 100 250 500 Chlormadinone acetate 5 10 20 50 100 250 500 Fluoxymesterone 10 20 40 100 200 500 1000 Danazol 10 20 40 100 200 500 1000 1.3.2. Pretreatment of sample Milk samples were pretreated with reference to GB/T 21981-2008 "Hormone Multi-Residue Detection Method for Animal-derived Food - LC-MS Method". ■Results and discussion 2.1. Q1 Scan and Product Ion Scan Mass Spectra of the Hormone Standards 1. The Q1 scan mass spectrum (left) and Product Ion Scan mass spectrum (CE value was -22V) (right) of androstenedione. No. LCMSMS-065E 2. The Q1 scan mass spectrum (left) and Product Ion Scan mass spectrum (CE value was -21V) (right) of boldenone. 3. The Q1 scan mass spectrum (left) and Product Ion Scan mass spectrum (CE value was -40V) (right) of danazol. 4. The Q1 scan mass spectrum (left) and Product Ion Scan mass spectrum (CE value was -32V) (right) of fluoxymesterone. No. LCMSMS-065E 5. The Q1 scan mass spectrum (left) and Product Ion Scan mass spectrum (CE value was -25V) (right) of testosterone. 6. The Q1 scan mass spectrum (left) and Product Ion Scan mass spectrum (CE value was -26V) (right) of methyltestosterone. 7. The Q1 scan mass spectrum (left) and Product Ion Scan mass spectrum (CE value was -18V) (right) of methandienone. No. LCMSMS-065E 8. The Q1 scan mass spectrum (left) and Product Ion Scan mass spectrum (CE value was -24V) (right) of nandrolone. 9. The Q1 scan mass spectrum (left) and Product Ion Scan mass spectrum (CE value was -25V) (right) of norandrostenedione. 10. The Q1 scan mass spectrum (left) and Product Ion Scan mass spectrum (CE value was -30V) (right) of trenbolone. No. LCMSMS-065E 11. The Q1 scan mass spectrum (left) and Product Ion Scan mass spectrum (CE value was -19V) (right) of megestrol acetate. 12. The Q1 scan mass spectrum (left) and Product Ion Scan mass spectrum (CE value was -29V) (right) of medroxyprogesterone.
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