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AbstractIn order to establish an HPLCMS/MS method for detecting progesterone in raw milk, raw milk was extracted with methanol, and then determined by HPLCMS/MS after the purification with a C18 solid phase extraction column. Qualitative determination was performed according to retention time and selective ion abundance ratio, and quantification was performed by external standard method. The results showed that progesterone had a good linear relation in the range of 0.1-5.0 ng/ml, with a correlation coefficient of 0.999 9. The detection limit of the established method was 0.02 μg/kg, and the quantification limit was 0.04 μg/kg. In the standard addition range of 0.1-5.0 ng/ml, the recovery ranged from 79.6% to 105.1%, with relative standard deviation≤8.9%. Compared with the industry standard, the detection limit and the quantification limit of the established method are significantly reduced, and the sensitivity of the method is enhanced, so this method is suitable for the detection of progesterone residue in fresh milk in pastures.
Key wordsHPLCMS/MS; Fresh and raw milk; Progesterone; Method; Establishment; Optimization
Received: July 18, 2018Accepted: November 3, 2018
Supported by Science and Technology Open Cooperation Project of Henan Province (162106000017); Science and Technology Peoplebenefiting Plan Project of Henan Province (152207110004); Major Science and Technology Program of Henan Province (16110051020); Puyang Science and Technology Plan Project (160215).
Zhen ZHANG (1979-), male, P. R. China, senior engineer, PhD, devoted to research about animal genetic breeding and reproduction.
*Corresponding author. Email: zzgxu@163.com.
Milk is known as "the most perfect food that nature gives to human beings" and is popular among the public. With the improvement of peoples living standards, the demand for milk has been increasing year by year, and has become a necessity for daily life［1］. At present, Chinas monitoring on the safety indicators of milk quality mainly focuses on microbes［2］, heavy metals［3］, illegal additives, pesticide residues［4］ and antibiotic residues［5］, while there have been few studies conducted on hormone residues in milk, especially fewer on progesterone and estradiol. At present, there is no official standard for hormone residues in milk in China［6］. With the deepening of research on environmental hormones, many scholars have begun to pay attention to the effects of hormones in food, especially in milk, on human health［7］. Milk contains more hormones, such as estrogen and progesterone, and the academic community is paying more and more attention to whether hormone levels in modern milk and longterm consumption of hormonecontaining milk by humans would adversely affect human health, especially infant health. The quality of raw milk is directly related to the quality and safety of dairy products, and the monitoring of hormone residues in raw milk from the source is of great significance to control of hormone residues in dairy products. Because cow breeds and feeding methods are quite different from those of 100 years ago［8-10］, and artificial insemination almost replaces natural mating, cows continue to lactate almost throughout the pregnancy, especially in late gestation, and many factors lead to continue increase of progesterone level in fresh milk［11］. About 75% of the commercially available milk comes from raw milk of pregnant cows［12］, so detecting progesterone residue in raw milk plays an important role in controlling progesterone level in raw milk from the source. In view of the shortcomings in the current industry standard: Determination of Progesterone Content in Milk by Liquid ChromatographyTandem Mass Spectrometry (NY/T 20692011), this study established and optimized the HPLCMS/MS detection of residual amount of progesterone in raw milk, aiming at improving the accuracy and sensitivity of the method for detecting progesterone residue in milk.
Materials
Tested raw milk
According to the Milk and Milk Products: Guidance on Sampling, fresh milk samples were collected from pastures close to the laboratory, stored at 4 ℃, and delivered to the laboratory within 2 h.
Main reagents
Progesterone standard (1 000 μg/ml, purity≥98.0%), purchased from Beijing Tanmo Quality Testing Technology Co., Ltd.; acetonitrile (HPLC≥99.9%), ammonium acetate (HPLC≥99.0%), purchased from Macklin; methanol (LCMS≥99.9%), formic acid (HPLC≥98.0%), purchased from Aladdin; lead acetate (analytical pure), purchased from Tianjin Kemiou Chemical Reagent Co., Ltd.; purified water, purchased from Watsons.
Main instruments
HPLC (LC1290), triple quadrupole mass spectrometer (MS/MS6460), solid phase extraction unit, column Eclipse Plus C18 (2.1 mm×100 mm, 1.8 μm), purchased from Agilent, USA; highspeed centrifuge, purchased from Sigma; ultrasonic instrument, purchased from Beijing Zhongshengming Technology Co., Ltd.; highspeed oscillatory mixer (QB600), purchased from Haimen Kylinbell Lab Instrument Co., Ltd.; C18 solid phase extraction column (500 mg, 6 ml), purchased from Agela company; 0.45 μm organic micropore filter membrane, purchased from Tianjin Jinteng Experiment Equipment Co., Ltd. Methods
LC and MS parameters
Setting of LC parameters
Mobile phase A was 5 mmol/L ammonium acetate aqueous solution (containing 0.1% formic acid), and mobile phase B was acetonitrile. Gradient elution was adopted and performed at a flow rate of 0.3 ml/min under an injection volume of 10 μl and a column temperature at 40 ℃.
Setting of MS parameters
The ion source was an electrospray ionization (ESI) source, and the scanning mode was positive ion scanning. The MS detection was performed at an ion spray voltage of 3.5 kV with nitrogen gas as drying gas at a flow rate of 10 L/min and a temperature of 350 ℃; and the collision gas was highpurity nitrogen, the cracking voltage was 130 V, and the scanning mode was Multiple Reaction Monitoring (MRM). The specific parameters were to be optimized.
Preparation of gradient standard solutions
A progesterone standard solution was accurately pipetted and diluted with methanol to a stock solution with a concentration of 100 ng/ml, which was then diluted to 0.1, 0.5, 1.0, 2.0 and 5.0 ng/ml standard working solutions, respectively.
Treatment of milk samples
Sample extraction
5.000 g of a certain amount of milk sample (accurate to 0.001 g) was accurately weighed and placed in a 50 ml centrifuge tube, into which 15 ml of methanol and 1 ml of 20% lead acetate solution were added. The sample was then extracted by steps of vortexmixing for 30 s, ultrasonically extracting for 10 min and centrifuging at 10 000 r/min for 10 min, obtaining a supernatant, which was added into a 25 ml volumetric flask. The residue was added with 5 ml of methanol, and extracted according to above extraction steps, i.e., vortexmixing for 30 s, ultrasonically extracting for 10 min and centrifuging at 10 000 r/min for 10 min. The supernatants were mixed and added with methanol to constant volume, obtaining a methanol extract.
Sample purification
The C18 solid phase extraction column was placed on a solid phase extraction apparatus and activated. The column was added with 5 ml of methanol and then washed with 5 ml of water. 5 ml of the methanol extract was diluted with 5 ml of water, obtaining 10 ml of mixture, which was added into the C18 solid phase extraction column. The column was then washed with 5 ml of water and eluted with 2 ml of acetonitrile. The eluate was filtered with a 0.55 μm organic filter for HPLCMS/MS analysis. Linear range
The calibration standard curve method was used in this test, and the external standard method was used for quantification. The standard curve was drawn with concentration as the abscissa and response value as the ordinate, and the curve equation and the correlation coefficient (R2) were obtained.
Detection limit and quantitation limit of the method
A recovery test was carried out to determine the detection limit and quantitation limit of the established method. A series of gradient progesterone standard solutions were added to blank pure milk containing no progesterone. During the detection of spiked pure milk, 5.000 g of fresh milk was detected for more than 10 times according to the method established in this study, and the detection limit and quantitation limit were the concentrations at the chromatographic signaltonoise ratios of 3 and 10, respectively［13］.
Recovery and precision
The recovery of the method was verified by adding the progesterone standard solution to blank sample and quantifying by external standard method. During the test, each addition level was detected repeatedly for 6 times, so as to verify the reliability of the method.
Results and Analysis
Optimization of LC conditions
The LC separation used an Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm) with A ( 5 mmol /L ammonium acetate solution + 0.1% formic acid) and B (acetonitrile) as the mobile phase for gradient elution (the gradient elution conditions are shown in Table 1) at a flow rate of 0.3 ml/min under a column temperature of 40 ℃. Under these conditions, progesterone showed good retention with a retention time of about 2.70 min (Fig. 1).
Table 1Gradient conditions
Elution time∥minA∥%B∥%Flow rate∥ml/min
090100.3
1.0035650.3
2.0035650.3
4.0001000.3
4.0190100.3
6.0090100.3
Note: The addition concentration was 0.5 ng/ml.
Fig. 1MRM chromatogram of spiked sample
Optimization of MS conditions
The 0.5 ng/ml progesterone standard solution was injected into the ion source by flow injection at a flow rate of 0.3 ml/min, and firstorder mass spectrometry was performed in the ESI source positive mode, obtaining a parent ion peak. The parent ion was then subjected to secondorder mass spectrometry, obtaining daughter ions and a secondorder mass spectrum. The cracking voltage and collision energy parameter were optimized to maximize the intensities of target parent ion and daughter ions, so as to determine the best MS parameters (Table 2). Table 2MRM parameters of progesterone
CompoundParention∥m/zDaughterion∥m/zCrackingvoltage∥VCollisionenergy∥V
Progesterone315108.913020
Progesterone31596.913024
Qualitative and quantitative detection was carried out according to the principle of "three ions". The daughter ion 315>108.9 was used for quantification, and the daughter ion 315>96.9 was used for qualitative detection. When the two ions are present in the sample at the same time, and the ion abundance (329.1>108.9 /329.1>96.9) is consistent with the standard solution of similar concentration (Table 3), it can be determined that the sample contains progesterone. It could be seen from Fig. 2 that in the concentration range of 0.1-5.0 ng/ml, the linear equation was y=2 448.1x-13.6, R2=0.999 9, indicating that progesterone has a good linear relation in the range of 0.1-5.0 ng/ml.
Detection limit and quantitation limit of the method
The detection limit of the established method was determined to be 0.02 μg/kg, and the quantification limit was 0.04 μg/kg, which satisfies the requirement for sensitivity of the detection of progesterone in raw milk.
Table 3Maximum allowable deviation of relative ion abundance during qualitative detection
Relative ion abundance＞50%20%-50%10%-20%＜10%
Maximum allowable deviation±20±25±30±50
Fig. 2Standard curve
Recovery and precision
The results are shown in Table 4.
It could be seen from Table 4 that in the concentration range of 0.1-1.0 ng/ml, the recovery of progesterone in the raw milk ranged from 79.6% to 105.1%, RSD≤8.9%, which satisfies the requirements for progesterone testing in raw milk.
Table 4Recovery and precision of progesterone added into raw milk
SampleAddition concentrationng/mlRecovery%Relative standarddeviation (RSD)∥%
Pure milk0.179.6-105.18.9
Pure milk0.589.2-100.74.1
Pure milk1.092.3-99.02.5
Agricultural Biotechnology2019
Discussion and Conclusions
Selection of elution conditions
The elution methods of LC column include gradient elution and isocratic elution. When isocratic elution is used, the composition of the mobile phase remains unchanged during the chromatographic separation process, and during gradient elution, the composition of the mobile phase changed linearly over time, i.e., a linear gradient. At present, LC columns mostly use gradient elution, because if samples contain impurities that can be firmly retained on columns, these impurities would remain on columns in the case of isocratic elution, which reduces the separation efficiency of columns and also interferes subsequent sample separation. These problems can be solved with gradient elution that completely removes impurities from the column using a highintensity mobile phase before the end of the program, so that the column can be maintained at the initial state for next injection. Compared with isocratic elution, gradient elution protects columns and improves column separation efficiency while improving column reproducibility. The LC conditions set in the progesterone industry standard by the Ministry of Agriculture: Determination of Progesterone Content in Milk by Liquid ChromatographyTandem Mass Spectrometry (NY/T 20692011) include using acetonitrile and 3 mmol/L ammonium acetate solution at a ratio of 65∶35 for isocratic elution. In this study, directing at the problems caused by isocratic elution, a gradient elution method was established to separate the progesterone in the sample, during which the proportions of 5 mmol/L ammonium acetate solution and acetonitrile changed over time. The principle of proportion setting is to start elution with a high proportion of aqueous phase, then gradually change to a high proportion of organic phase, and finally return to a high proportion of aqueous phase to wash the column. It could be seen from the chromatogram obtained by gradient elution that progesterone has a good retention effect at 2.70 min with good reproducibility.
Detection limit and quantitation limit
The current standard for LCMS/MS for the detection of progesterone in milk has a detection limit of 1 μg/kg. In this study, by changing LC conditions and optimizing MS conditions, the detection limit and the quantitation limit of the method for detecting progesterone were greatly reduced for establishing the method of detecting progesterone residue in raw milk. When weighing 5.000 g of raw milk, the concentrations at the signaltonoise ratios of 3∶1 and 10∶1 were selected as the detection limit and the quantification limit, respectively, obtaining the detection limit of 0.02 μg/kg and the quantification limit of 0.04 μg/kg. This signifi
Key wordsHPLCMS/MS; Fresh and raw milk; Progesterone; Method; Establishment; Optimization
Received: July 18, 2018Accepted: November 3, 2018
Supported by Science and Technology Open Cooperation Project of Henan Province (162106000017); Science and Technology Peoplebenefiting Plan Project of Henan Province (152207110004); Major Science and Technology Program of Henan Province (16110051020); Puyang Science and Technology Plan Project (160215).
Zhen ZHANG (1979-), male, P. R. China, senior engineer, PhD, devoted to research about animal genetic breeding and reproduction.
*Corresponding author. Email: zzgxu@163.com.
Milk is known as "the most perfect food that nature gives to human beings" and is popular among the public. With the improvement of peoples living standards, the demand for milk has been increasing year by year, and has become a necessity for daily life［1］. At present, Chinas monitoring on the safety indicators of milk quality mainly focuses on microbes［2］, heavy metals［3］, illegal additives, pesticide residues［4］ and antibiotic residues［5］, while there have been few studies conducted on hormone residues in milk, especially fewer on progesterone and estradiol. At present, there is no official standard for hormone residues in milk in China［6］. With the deepening of research on environmental hormones, many scholars have begun to pay attention to the effects of hormones in food, especially in milk, on human health［7］. Milk contains more hormones, such as estrogen and progesterone, and the academic community is paying more and more attention to whether hormone levels in modern milk and longterm consumption of hormonecontaining milk by humans would adversely affect human health, especially infant health. The quality of raw milk is directly related to the quality and safety of dairy products, and the monitoring of hormone residues in raw milk from the source is of great significance to control of hormone residues in dairy products. Because cow breeds and feeding methods are quite different from those of 100 years ago［8-10］, and artificial insemination almost replaces natural mating, cows continue to lactate almost throughout the pregnancy, especially in late gestation, and many factors lead to continue increase of progesterone level in fresh milk［11］. About 75% of the commercially available milk comes from raw milk of pregnant cows［12］, so detecting progesterone residue in raw milk plays an important role in controlling progesterone level in raw milk from the source. In view of the shortcomings in the current industry standard: Determination of Progesterone Content in Milk by Liquid ChromatographyTandem Mass Spectrometry (NY/T 20692011), this study established and optimized the HPLCMS/MS detection of residual amount of progesterone in raw milk, aiming at improving the accuracy and sensitivity of the method for detecting progesterone residue in milk.
Materials
Tested raw milk
According to the Milk and Milk Products: Guidance on Sampling, fresh milk samples were collected from pastures close to the laboratory, stored at 4 ℃, and delivered to the laboratory within 2 h.
Main reagents
Progesterone standard (1 000 μg/ml, purity≥98.0%), purchased from Beijing Tanmo Quality Testing Technology Co., Ltd.; acetonitrile (HPLC≥99.9%), ammonium acetate (HPLC≥99.0%), purchased from Macklin; methanol (LCMS≥99.9%), formic acid (HPLC≥98.0%), purchased from Aladdin; lead acetate (analytical pure), purchased from Tianjin Kemiou Chemical Reagent Co., Ltd.; purified water, purchased from Watsons.
Main instruments
HPLC (LC1290), triple quadrupole mass spectrometer (MS/MS6460), solid phase extraction unit, column Eclipse Plus C18 (2.1 mm×100 mm, 1.8 μm), purchased from Agilent, USA; highspeed centrifuge, purchased from Sigma; ultrasonic instrument, purchased from Beijing Zhongshengming Technology Co., Ltd.; highspeed oscillatory mixer (QB600), purchased from Haimen Kylinbell Lab Instrument Co., Ltd.; C18 solid phase extraction column (500 mg, 6 ml), purchased from Agela company; 0.45 μm organic micropore filter membrane, purchased from Tianjin Jinteng Experiment Equipment Co., Ltd. Methods
LC and MS parameters
Setting of LC parameters
Mobile phase A was 5 mmol/L ammonium acetate aqueous solution (containing 0.1% formic acid), and mobile phase B was acetonitrile. Gradient elution was adopted and performed at a flow rate of 0.3 ml/min under an injection volume of 10 μl and a column temperature at 40 ℃.
Setting of MS parameters
The ion source was an electrospray ionization (ESI) source, and the scanning mode was positive ion scanning. The MS detection was performed at an ion spray voltage of 3.5 kV with nitrogen gas as drying gas at a flow rate of 10 L/min and a temperature of 350 ℃; and the collision gas was highpurity nitrogen, the cracking voltage was 130 V, and the scanning mode was Multiple Reaction Monitoring (MRM). The specific parameters were to be optimized.
Preparation of gradient standard solutions
A progesterone standard solution was accurately pipetted and diluted with methanol to a stock solution with a concentration of 100 ng/ml, which was then diluted to 0.1, 0.5, 1.0, 2.0 and 5.0 ng/ml standard working solutions, respectively.
Treatment of milk samples
Sample extraction
5.000 g of a certain amount of milk sample (accurate to 0.001 g) was accurately weighed and placed in a 50 ml centrifuge tube, into which 15 ml of methanol and 1 ml of 20% lead acetate solution were added. The sample was then extracted by steps of vortexmixing for 30 s, ultrasonically extracting for 10 min and centrifuging at 10 000 r/min for 10 min, obtaining a supernatant, which was added into a 25 ml volumetric flask. The residue was added with 5 ml of methanol, and extracted according to above extraction steps, i.e., vortexmixing for 30 s, ultrasonically extracting for 10 min and centrifuging at 10 000 r/min for 10 min. The supernatants were mixed and added with methanol to constant volume, obtaining a methanol extract.
Sample purification
The C18 solid phase extraction column was placed on a solid phase extraction apparatus and activated. The column was added with 5 ml of methanol and then washed with 5 ml of water. 5 ml of the methanol extract was diluted with 5 ml of water, obtaining 10 ml of mixture, which was added into the C18 solid phase extraction column. The column was then washed with 5 ml of water and eluted with 2 ml of acetonitrile. The eluate was filtered with a 0.55 μm organic filter for HPLCMS/MS analysis. Linear range
The calibration standard curve method was used in this test, and the external standard method was used for quantification. The standard curve was drawn with concentration as the abscissa and response value as the ordinate, and the curve equation and the correlation coefficient (R2) were obtained.
Detection limit and quantitation limit of the method
A recovery test was carried out to determine the detection limit and quantitation limit of the established method. A series of gradient progesterone standard solutions were added to blank pure milk containing no progesterone. During the detection of spiked pure milk, 5.000 g of fresh milk was detected for more than 10 times according to the method established in this study, and the detection limit and quantitation limit were the concentrations at the chromatographic signaltonoise ratios of 3 and 10, respectively［13］.
Recovery and precision
The recovery of the method was verified by adding the progesterone standard solution to blank sample and quantifying by external standard method. During the test, each addition level was detected repeatedly for 6 times, so as to verify the reliability of the method.
Results and Analysis
Optimization of LC conditions
The LC separation used an Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm) with A ( 5 mmol /L ammonium acetate solution + 0.1% formic acid) and B (acetonitrile) as the mobile phase for gradient elution (the gradient elution conditions are shown in Table 1) at a flow rate of 0.3 ml/min under a column temperature of 40 ℃. Under these conditions, progesterone showed good retention with a retention time of about 2.70 min (Fig. 1).
Table 1Gradient conditions
Elution time∥minA∥%B∥%Flow rate∥ml/min
090100.3
1.0035650.3
2.0035650.3
4.0001000.3
4.0190100.3
6.0090100.3
Note: The addition concentration was 0.5 ng/ml.
Fig. 1MRM chromatogram of spiked sample
Optimization of MS conditions
The 0.5 ng/ml progesterone standard solution was injected into the ion source by flow injection at a flow rate of 0.3 ml/min, and firstorder mass spectrometry was performed in the ESI source positive mode, obtaining a parent ion peak. The parent ion was then subjected to secondorder mass spectrometry, obtaining daughter ions and a secondorder mass spectrum. The cracking voltage and collision energy parameter were optimized to maximize the intensities of target parent ion and daughter ions, so as to determine the best MS parameters (Table 2). Table 2MRM parameters of progesterone
CompoundParention∥m/zDaughterion∥m/zCrackingvoltage∥VCollisionenergy∥V
Progesterone315108.913020
Progesterone31596.913024
Qualitative and quantitative detection was carried out according to the principle of "three ions". The daughter ion 315>108.9 was used for quantification, and the daughter ion 315>96.9 was used for qualitative detection. When the two ions are present in the sample at the same time, and the ion abundance (329.1>108.9 /329.1>96.9) is consistent with the standard solution of similar concentration (Table 3), it can be determined that the sample contains progesterone. It could be seen from Fig. 2 that in the concentration range of 0.1-5.0 ng/ml, the linear equation was y=2 448.1x-13.6, R2=0.999 9, indicating that progesterone has a good linear relation in the range of 0.1-5.0 ng/ml.
Detection limit and quantitation limit of the method
The detection limit of the established method was determined to be 0.02 μg/kg, and the quantification limit was 0.04 μg/kg, which satisfies the requirement for sensitivity of the detection of progesterone in raw milk.
Table 3Maximum allowable deviation of relative ion abundance during qualitative detection
Relative ion abundance＞50%20%-50%10%-20%＜10%
Maximum allowable deviation±20±25±30±50
Fig. 2Standard curve
Recovery and precision
The results are shown in Table 4.
It could be seen from Table 4 that in the concentration range of 0.1-1.0 ng/ml, the recovery of progesterone in the raw milk ranged from 79.6% to 105.1%, RSD≤8.9%, which satisfies the requirements for progesterone testing in raw milk.
Table 4Recovery and precision of progesterone added into raw milk
SampleAddition concentrationng/mlRecovery%Relative standarddeviation (RSD)∥%
Pure milk0.179.6-105.18.9
Pure milk0.589.2-100.74.1
Pure milk1.092.3-99.02.5
Agricultural Biotechnology2019
Discussion and Conclusions
Selection of elution conditions
The elution methods of LC column include gradient elution and isocratic elution. When isocratic elution is used, the composition of the mobile phase remains unchanged during the chromatographic separation process, and during gradient elution, the composition of the mobile phase changed linearly over time, i.e., a linear gradient. At present, LC columns mostly use gradient elution, because if samples contain impurities that can be firmly retained on columns, these impurities would remain on columns in the case of isocratic elution, which reduces the separation efficiency of columns and also interferes subsequent sample separation. These problems can be solved with gradient elution that completely removes impurities from the column using a highintensity mobile phase before the end of the program, so that the column can be maintained at the initial state for next injection. Compared with isocratic elution, gradient elution protects columns and improves column separation efficiency while improving column reproducibility. The LC conditions set in the progesterone industry standard by the Ministry of Agriculture: Determination of Progesterone Content in Milk by Liquid ChromatographyTandem Mass Spectrometry (NY/T 20692011) include using acetonitrile and 3 mmol/L ammonium acetate solution at a ratio of 65∶35 for isocratic elution. In this study, directing at the problems caused by isocratic elution, a gradient elution method was established to separate the progesterone in the sample, during which the proportions of 5 mmol/L ammonium acetate solution and acetonitrile changed over time. The principle of proportion setting is to start elution with a high proportion of aqueous phase, then gradually change to a high proportion of organic phase, and finally return to a high proportion of aqueous phase to wash the column. It could be seen from the chromatogram obtained by gradient elution that progesterone has a good retention effect at 2.70 min with good reproducibility.
Detection limit and quantitation limit
The current standard for LCMS/MS for the detection of progesterone in milk has a detection limit of 1 μg/kg. In this study, by changing LC conditions and optimizing MS conditions, the detection limit and the quantitation limit of the method for detecting progesterone were greatly reduced for establishing the method of detecting progesterone residue in raw milk. When weighing 5.000 g of raw milk, the concentrations at the signaltonoise ratios of 3∶1 and 10∶1 were selected as the detection limit and the quantification limit, respectively, obtaining the detection limit of 0.02 μg/kg and the quantification limit of 0.04 μg/kg. This signifi