Ultrasonic-Assisted Pretreatment Method for Extraction of Multiple Steroid Hormones in Sediment

Abstract

The disclosure discloses an ultrasonic-assisted pretreatment method for extraction of multiple steroid hormones in a sediment, including the following steps: (1) lyophilizing the sediment, grinding the sediment, and passing the ground sediment through a 40-60-mesh sieve; (2) placing the sample obtained in step (1) in a container; (3) adding an extractant to the container in step (2), shaking the mixture for 15 s-30 s, centrifuging the mixture to collect an supernatant after ultrasonication, and repeating extraction three times; where the extractants used in the three times of extraction are two of methanol, acetonitrile and acetone; and (4) after mixing the supernatants of the three times of extraction obtained in step (3), concentrating the mixture under a nitrogen flow at 20-30° C., passing the concentrated mixture through a filter, and performing detection. By using the method of the disclosure, the maximum recovery can be up to 100%. The application range is wide, and a recovery of the plurality of steroid hormones each can be up to 73% or above.

Claims

1. A pretreatment method for extraction of multiple steroid hormones in a sediment, comprising the following steps: (1) lyophilizing the sediment, and grinding the sediment to 40-60 mesh; (2) placing a sample obtained in step (1) in a container; (3) adding an extractant to the container in step (2) for extraction, shaking a mixture for 15-30 s, centrifuging the mixture to collect a supernatant after ultrasonication, and repeating the extraction three times; and (4) after mixing the supernatants of the three times of extraction obtained in step (3), concentrating the mixture under a nitrogen flow at 20-30° C., and passing the concentrated mixture through a filter; wherein solvents for the three times of extraction are methanol, acetonitrile and acetonitrile, respectively, or methanol, methanol and acetone, respectively, or methanol, acetone and acetone, respectively; and the pretreatment method does not need an SPE cleaning step.

2. The pretreatment method according to claim 1, wherein the steroid hormones comprise estrogens, androgens, and progestogens.

3. The pretreatment method according to claim 1, wherein in the step (1), the lyophilizing is to pre-freeze the sediment at −60 to −80° C. overnight and lyophilize the sediment at −45° C.

4. The pretreatment method according to claim 1, wherein in the step (2), an amount of the sample placed in the container is 0.5-5.0 g.

5. The pretreatment method according to claim 1, wherein in the step (2), the container is made of a polytetrafluoroethylene material.

6. The pretreatment method according to claim 4, wherein in the step (3), a volume of the extractant is 5-10 mL.

7. The pretreatment method according to claim 1, wherein in the step (3), a frequency of ultrasound in the ultrasonication is 45-60 kHz.

8. A method for detecting a content of steroid hormones in a sediment or sandy soil, comprising: treating a sample by using the pretreatment method according to claim 1 to extract the steroid hormones in the sample; and then analyzing the sample obtained by the extraction by high performance liquid chromatography-tandem mass spectrometry to quantitatively detect the content of the steroid hormones in the sample, wherein the steroid hormones comprise estrogens, androgens and progestogens; and the sample is the sediment or sandy soil.

9. The method according to claim 8, wherein conditions for the liquid chromatography when analyzing the estrogens are that: a mobile phase A is 0.05% ammonia water, a mobile phase B is acetonitrile, a flow rate is 0.2 mL/min, an injection volume is 5 μL, and a column temperature is 40° C.; and conditions for elution are that: at 0-0.25 min, a mobile phase is 70% mobile phase A+30% mobile phase B; at 0.25-3 min, the mobile phase B is gradually increased to 90%, and a condition is held for 2 min; and then, a column is immediately restored to an initial mobile phase, namely 70% mobile phase A+30% mobile phase B, and a condition is held for 1 min.

10. The method according to claim 9, wherein conditions for the mass spectrometry when analyzing the estrogens are that: electrospray ionization is used in negative ion mode (BO; a scanning mode is multiselected reaction monitoring (MRM); a capillary voltage is 2.5 kV; a cone voltage and an extractor voltage are 30.0 V and 3.0 V, respectively; an ion source temperature and a desolvation temperature are 150° C. and 350° C., respectively; a cone gas flow and a desolvation gas flow are 50 L/hr and 700 L/hr, respectively; and a collision gas flow is 0.16 mL/min.

11. The method according to claim 8, wherein conditions for the chromatography when analyzing the androgens and the progestogens are that: a mobile phase A is a methanol solution containing 0.1% of formic acid, a volume ratio of water to methanol in the methanol solution is 98:2, a mobile phase B is acetonitrile, a flow rate is 0.4 mL/min, an injection volume is 5 μL, and a column temperature is 40° C.; and conditions for elution are that: at 0-0.25 min, a mobile phase is 90% mobile phase A+10% mobile phase B; at 0.25-1 min, the mobile phase B is gradually increased to 70%; at 1-3 min, the mobile phase B is increased to 95%, and a condition is held for 1 min; and then, a column is immediately restored to an initial mobile phase, namely 90% mobile phase A+10% mobile phase B, and a condition is held for 1 min.

12. The method according to claim 11, wherein when detecting the androgens and the progestogens, electrospray ionization is used in positive ion mode (ESI.sup.+), and a scanning mode is multiselected reaction monitoring (MRM).

13. The method according to claim 12, wherein conditions for the mass spectrometry are that: a capillary voltage is 3.0 kV; a cone voltage and an extractor voltage are 30.0 V and 3.0 V, respectively; an ion source temperature and a desolvation temperature are 150° C. and 500° C., respectively; a cone gas flow and a desolvation gas flow are 50 L/hr and 900 L/hr, respectively; and a collision gas flow is 0.16 mL/min.

14. The method according to claim 8, wherein the steroid hormones comprise one or more of estrone, estradiol, ethinylestradiol, androstenedione, testosterone, methyltestosterone, levonorgestrel and progesterone.

Description

BRIEF DESCRIPTION OF FIGURES

[0034] FIG. 1 is a flow chart of ultrasonic-assisted extraction of multiple steroid hormones in a sediment.

[0035] FIG. 2 is a diagram of recoveries of steroid hormones by using different extractant combinations, where E1 is estrone, E2 is estradiol (17β-estradiol), EE2 is ethinylestradiol, AND is androstenedione, TES is testosterone, MET is methyltestosterone (17α-methyltestosterone), LEV is levonorgestrel, and PRO is progesterone.

DETAILED DESCRIPTION

[0036] Embodiments of the disclosure will be described below with specific examples.

[0037] The disclosure discloses an ultrasonic-assisted pretreatment method for extraction of multiple residual steroid hormones in a sediment, which can be used to extract 8 steroid hormones such as estrogens, androgens and progestogens in the sediment. The method uses the following instruments and reagents:

[0038] Instruments: ultrasonic instrument (Ningbo Scientz Biotechnology Co., Ltd., SB-500DTY); lyophilizer (LABCONCO); centrifuge (Eppendorf, Centrifuge 5804 R); vortex shaker (Shanghai Huxi Analysis Instrument Factory Co., Ltd., WH-1 Mini Vortex Mixer); nitrogen purging device (Reeko Instrument USA, AUTO SPE-06D); and high performance liquid chromatography-tandem mass spectrometry (Waters, ACQUITY UPLC Xevo TQ), Waters ACQUITY UPLC R BEH C18 (100 mm×2.1 mm×1.7 μm).

[0039] Reagents: the extraction solvents include methanol, acetonitrile and acetone, all of which are of HPLC grade (Merck, Germany), and ultrapure water.

[0040] The test method of the recovery is as follows:

[0041] 100 μL of a mixed standard solution of steroid hormones with a concentration of 1 mg/L is added to the sediment or sandy soil, that is, the spiking amount is 100 ng. At the same time, unspiked blanks are prepared. Two duplicate samples are prepared for each experimental group. After 2 hours of standing, extraction and detection are performed, and the recovery is calculated according to the following formula:

[00001]$R=\frac{M-M_0}{100}\times 100.Math.\%$

[0042] where R is the recovery, %; M is the detected amount of the spiked sample, ng; and Mo is the detected amount of the blank sample, ng.

Example 1: Establishment of Standard Curves

[0043] (1) Firstly, each steroid hormone standard was accurately prepared into a single-solute standard stock solution with a concentration of 1 g/L, the single-solute standard stock solutions were respectively diluted into 1 mg/L standard samples, and the standard samples were subjected to LC-MS/MS analysis to determine the respective retention times.

[0044] Conditions for the liquid chromatography when analyzing the estrogens were: a mobile phase A was 0.05% ammonia water, a mobile phase B was acetonitrile, a flow rate was 0.2 mL/min, an injection volume was 5 μL, and a column temperature was 40° C. Conditions for elution were: at 0-0.25 min, the mobile phase was 70% phase A+30% phase B; at 0.25-3 min, the phase B was gradually increased to 90%, and the condition was held for 2 min; and then, the column was immediately restored to the initial mobile phase, namely 70% phase A+30% phase B, and the condition was held for 1 min.

[0045] Conditions for the mass spectrometry were: electrospray ionization was used in negative ion mode (ESI.sup.−); a scanning mode was multiselected reaction monitoring (MRM); a capillary voltage was 2.5 kV; a cone voltage and an extractor voltage were respectively 30.0 V and 3.0 V; an ion source temperature and a desolvation temperature were respectively 150° C. and 350° C.; a cone gas flow and a desolvation gas flow were respectively 50 L/hr and 700 L/hr; and a collision gas flow was 0.16 mL/min.

[0046] Conditions for the chromatography when analyzing the androgens and the progestogens were: a mobile phase A was a methanol solution containing 0.1% of formic acid (a ratio of water to methanol was 98:2, v/v), a mobile phase B was acetonitrile, a flow rate was 0.4 mL/min, an injection volume was 5 μL, and a column temperature was 40° C. Conditions for elution were: at 0-0.25 min, the mobile phase was 90% phase A+10% phase B; at 0.25-1 min, the phase B was gradually increased to 70%; at 1-3 min, the phase B was increased to 95%, and the condition was held for 1 min; and then, the column was immediately restored to the initial mobile phase, namely 90% phase A+10% phase B, and the condition was held for 1 min.

[0047] Conditions for the mass spectrometry were: when detecting the androgens and the progestogens, electrospray ionization is used in positive ion mode (ESI.sup.+); a scanning mode was multiselected reaction monitoring (MRM); a capillary voltage was 3.0 kV; a cone voltage and an extractor voltage were respectively 30.0 V and 3.0 V; an ion source temperature and a desolvation temperature were respectively 150° C. and 500° C.; a cone gas flow and a desolvation gas flow were respectively 50 L/hr and 900 L/hr; and a collision gas flow was 0.16 mL/min.

[0048] (2) Then, mixed standard stock solutions of steroid hormones with a concentration of 10 mg/L were prepared, and diluted into a series of mixed standard solutions with concentrations of 0.5 μg/L, 2 μg/L, 5 μg/L, 20 μg/L, 50 μg/L and 200 μg/L respectively. The concentrations of the steroid hormones in the mixed standard solutions were uniform. Finally, by taking a nominal injection concentration as the abscissa X (μg/L) and a peak area as the ordinate Y, the standard curves and detection limits of corresponding steroid hormones were obtained. The results are shown in Table 1.

TABLE-US-00001 TABLE 1 Degree Retention of Limit of Linear time fitting quantitation range Steroid hormone (min) Standard curve (R.sup.2) (ppb) (ppb) Estrone E1 3.29 Y = 308.994X − 19.467 0.9980 0.292 0.5-200 Estradiol E2 3.02 Y = 52.486X − 7.115 0.9984 0.381 0.5-200 Ethinylestradiol 3.17 Y = 40.920X − 6.289 0.9960 0.710 0.5-200 EE2 Androstenedione 1.88 Y = 1712.42X − 239.601 0.9933 0.110 0.5-200 AND Testosterone TES 1.80 Y = 1366.26X − 132.456 0.9967 0.222 0.5-200 Methyltestosterone 1.87 Y = 1130.24X + 42.805 0.9981 0.147 0.5-200 MET Levonorgestrel 1.93 Y = 93.07X − 15.607 0.9933 0.444 2.0-200 LEV Progesterone PRO 2.21 Y = 1894.85X − 40.546 0.9968 0.270 0.5-200

Example 2: An Ultrasonic-Assisted Pretreatment Method Tor Extraction of Multiple Steroid Hormones in Sediment

[0049] As shown in FIG. 1, a collected sediment was uniformly spread on a glass petri dish with a thickness of not more than 5 mm. The sediment was placed in a −80° C. refrigerator overnight. The sediment was taken out the next day, immediately placed in a lyophilizer and lyophilized. After 16-24 hours, the sediment was taken out, ground in a mortar, and passed through a 50-mesh sieve for later extraction. The sample that could not be subjected to extraction immediately was wrapped with aluminum foil, sealed in a sealing bag, and stored in a −20° C. refrigerator.

[0050] 2.0 g of the lyophilized and ground sediment was accurately weighed, and placed in a 50 mL polytetrafluoroethylene centrifuge tube. 5 mL of methanol was added. After the centrifuge tube was well capped, the mixture was mixed thoroughly with a vortex shaker, subjected to ultrasonic extraction under the conditions of 20° C. and 59 kHz for 15 min, and then centrifuged in a centrifuge at 4000 rpm for 30 min for solid-liquid separation. The supernatant was sucked out with a glass pipette and put in a glass nitrogen purging tube. The above steps are repeated 2 times, but the extraction solvent was changed to 5 mL of acetonitrile. When mixing with the vortex shaker, it must be ensured that the sediment after centrifugal separation could be fully resuspended in the extractant without lumpy solids. About 15 mL of the supernatant after three times of centrifugation was collected in a nitrogen purging tube, concentrated to 1 mL under a steady nitrogen flow, passed through a 0.22-μm PTFE filter, and stored in an amber autosampler vial for detection.

[0051] The extract was analyzed by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Conditions for the liquid chromatography when analyzing the estrogens were: a mobile phase A was 0.05% ammonia water, a mobile phase B was acetonitrile, a flow rate was 0.2 mL/min, an injection volume was 5 μL, and a column temperature was 40° C. Conditions for elution were: at 0-0.25 min, the mobile phase was 70% phase A+30% phase B; at 0.25-3 min, the phase B was gradually increased to 90%, and the condition was held for 2 min; and then, the column was immediately restored to the initial mobile phase, namely 70% phase A+30% phase B, and the condition was held for 1 min. Conditions for the mass spectrometry were: electrospray ionization was used in negative ion mode (ESI.sup.−); a scanning mode was multiselected reaction monitoring (MRM); a capillary voltage was 2.5 kV; a cone voltage and an extractor voltage were respectively 30.0 V and 3.0 V; an ion source temperature and a desolvation temperature were respectively 150° C. and 350° C.; a cone gas flow and a desolvation gas flow were respectively 50 L/hr and 700 L/hr; and a collision gas flow was 0.16 mL/min. Conditions for the chromatography when analyzing the androgens and the progestogens were: a mobile phase A was a methanol solution containing 0.1% of formic acid (a ratio of water to methanol was 98:2, v/v), a mobile phase B was acetonitrile, a flow rate was 0.4 mL/min, an injection volume was 5 μL, and a column temperature was 40° C. Conditions for elution were: at 0-0.25 min, the mobile phase was 90% phase A+10% phase B; at 0.25-1 min, the phase B was gradually increased to 70%; at 1-3 min, the phase B was increased to 95%, and the condition was held for 1 min; and then, the column was immediately restored to the initial mobile phase, namely 90% phase A+10% phase B, and the condition was held for 1 min. Conditions for the mass spectrometry were: when detecting the androgens and the progestogens, electrospray ionization was used in positive ion mode (ESI.sup.+); a scanning mode was multiselected reaction monitoring (MRM); a capillary voltage was 3.0 kV; a cone voltage and an extractor voltage were respectively 30.0 V and 3.0 V; an ion source temperature and a desolvation temperature were respectively 150° C. and 500° C.; a cone gas flow and a desolvation gas flow were respectively 50 L/hr and 900 L/hr; and a collision gas flow was 0.16 mL/min.

Example 3: Pretreatment Method for Ultrasonic Assisted Extraction of Multiple Steroid Hormones in Sandy Soil

[0052] Sandy soil was placed on a glass petri dish with a thickness of not more than 5 mm. The sandy soil was placed in a −80° C. refrigerator overnight. The sediment was taken out the next day, immediately placed in a lyophilizer and lyophilized. After 16-24 hours, the sandy soil was taken out, ground in a mortar, and passed through a 50-mesh sieve for later extraction.

[0053] 2.0 g of the lyophilized sandy soil was accurately weighed, and placed in a 50 mL polytetrafluoroethylene centrifuge tube. 5 mL of methanol was added. After the centrifuge tube was well capped, the mixture was mixed thoroughly with a vortex shaker, subjected to ultrasonic extraction under the conditions of 30° C. and 59 kHz for 15 min, and then centrifuged in a centrifuge at 4000 rpm for 30 min for solid-liquid separation. The supernatant was sucked out with a glass pipette and put in a glass nitrogen purging tube. The above steps are repeated 2 times, but the extraction solvent was changed to 5 mL of acetonitrile. When mixing with the vortex shaker, it must be ensured that the sediment after centrifugal separation could be fully resuspended in the extractant without lumpy solids. About 15 mL of the supernatant after three times of centrifugation was collected in a nitrogen purging tube, concentrated to 1 mL under a steady nitrogen flow, passed through a 0.22-μm PTFE filter, and stored in an amber autosampler vial for detection.

[0054] The extract was analyzed by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Conditions for the liquid chromatography when analyzing the estrogens were: a mobile phase A was 0.05% ammonia water, a mobile phase B was acetonitrile, a flow rate was 0.2 mL/min, an injection volume was 5 μL, and a column temperature was 40° C. Conditions for elution were: at 0-0.25 min, the mobile phase was 70% phase A+30% phase B; at 0.25-3 min, the phase B was gradually increased to 90%, and the condition was held for 2 min; and then, the column was immediately restored to the initial mobile phase, namely 70% phase A+30% phase B, and the condition was held for 1 min. Conditions for the mass spectrometry were: electrospray ionization was used in negative ion mode (ESI.sup.−); a scanning mode was multiselected reaction monitoring (MRM); a capillary voltage was 2.5 kV; a cone voltage and an extractor voltage were respectively 30.0 V and 3.0 V; an ion source temperature and a desolvation temperature were respectively 150° C. and 350° C.; a cone gas flow and a desolvation gas flow were respectively 50 L/hr and 700 L/hr; and a collision gas flow was 0.16 mL/min. Conditions for the chromatography when analyzing the androgens and the progestogens were: a mobile phase A was a methanol solution containing 0.1% of formic acid (a ratio of water to methanol was 98:2, v/v), a mobile phase B was acetonitrile, a flow rate was 0.4 mL/min, an injection volume was 5 μL, and a column temperature was 40° C. Conditions for elution were: at 0-0.25 min, the mobile phase was 90% phase A+10% phase B; at 0.25-1 min, the phase B was gradually increased to 70%; at 1-3 min, the phase B was increased to 95%, and the condition was held for 1 min; and then, the column was immediately restored to the initial mobile phase, namely 90% phase A+10% phase B, and the condition was held for 1 min. Conditions for the mass spectrometry were: when detecting the androgens and the progestogens, electrospray ionization was used in positive ion mode (ESI.sup.+); a scanning mode was multiselected reaction monitoring (MRM); a capillary voltage was 3.0 kV; a cone voltage and an extractor voltage were respectively 30.0 V and 3.0 V; an ion source temperature and a desolvation temperature were respectively 150° C. and 500° C.; a cone gas flow and a desolvation gas flow were respectively 50 L/hr and 900 L/hr; and a collision gas flow was 0.16 mL/min.

Example 4

[0055] The solvent for the second extraction was changed to methanol, and the solvent for the third extraction was changed to acetone. The rest of the operations and steps were the same as in Example 2. That is, 5 mL of methanol, 5 mL of methanol and 5 mL of acetone were sequentially used for ultrasonic-assisted extraction of steroid hormones in the sediment. The results are shown in Table 2 and FIG. 2. The recoveries of the steroid hormones are 86%-106%.

Example 5

[0056] The solvent for the second extraction and the third extraction was changed to acetone. The rest of the operations and steps were the same as in Example 2. That is, 5 mL of methanol, 5 mL of acetone and 5 mL of acetone were sequentially used for ultrasonic-assisted extraction of steroid hormones in the sediment. The results are shown in Table 2 and FIG. 2. The recoveries of the steroid hormones are 73%-92%.

Example 6: Accuracy of Method

[0057] Recovery test: the sediment or sandy soil was spiked with 100 μL of a mixed standard solution of steroid hormones with each compound at 1 mg/L was added to the sediment or sandy soil, that is, the spiking amount was 100 ng. At the same time, unspiked blanks were prepared. Two duplicate samples were prepared for each experimental group. After 2 hours of standing, extraction and detection were performed, and the recovery of the method was calculated. The specific results are shown in Table 2.

Comparative Example 1

[0058] The solvent for the three times of extraction was changed to methanol, and the rest of operations and steps were the same as in Example 2. The results are shown in Table 2 and FIG. 2. The recoveries of the steroid hormones are 44%-50%, which are significantly lower than the recoveries 88%-116% in Example 2. When 5 mL of methanol is used for the three times of ultrasonic-assisted extraction on the steroid hormones in the sediment, the effect is not as good as that of the solvent combination of 5 mL of methanol, 5 mL of acetonitrile and 5 mL of acetonitrile, indicating that the recovery of extraction with one extractant is not as good as that of two extractants.

Comparative Example 2

[0059] After the supernatant obtained after the three times of extraction was concentrated to obtain 1 mL of sample, 250 mL of ultrapure water was added and completely mixed. Then, the mixture was passed at a flow rate of 10 mL/min through an Oasis HLB solid-phase extraction column (Waters, 6 cc, 500 mg) sequentially activated with 5 mL of methanol and 5 mL of ultrapure water. After the completion of the sample loading, impurities in the column were washed off with a 5% methanol solution, and the HLB column was blow-dried with nitrogen for 20 min. Then, the target analyte was sequentially eluted with 7 mL of methanol and 7 mL of a methanol-acetone solution (1:1, v/v), finally concentrated to 1 mL under a steady nitrogen flow, and stored in an amber autosampler vial for detection. The rest of operations and steps were the same as in Example 2. The results are shown in Table 2 and FIG. 2. The recoveries of the steroid hormones are 27%-33%, which are significantly lower than those in Example 2, indicating that the additional SPE cleaning step will result in a decrease in the recovery.

TABLE-US-00002 TABLE 2 Influence of different pretreatment methods on recoveries of steroid hormones Example Example Example Example Comparative Comparative Recovery/% 2 3 4 5 example 1 example 2 Estrone 96 — 92 81 — — Estradiol 88 98 86 73 48 33 Ethinylestradiol 111 110 103 92 49 33 Androstenedione 116 83 106 86 48 30 Testosterone 100 77 96 78 46 28 Methyltestosterone 99 76 94 78 44 27 Levonorgestrel 97 82 92 80 50 33 Progesterone 101 69 94 83 48 28 Note: Due to the lack of experimental standards, the recoveries of E1 in Example 2, Comparative examples 1 and 2 were not available in the disclosure.