Norethindrone, levonorgestrel, chlormadinone acetate and mifepristone were obtained from Beijing Century-Aoke Biotechnology Co. Ltd. (Beijing, China). Methanol, acetonitrile and formic acid, all of HPLC grade, were products from Merck KGaA (Darmstadt, Germany). Ammonium acetate, also HPLC grade, was from Kemiou Chemical Reagent Co. Ltd. (Tianjin, China), and ultra-pure water was the efflux out of a PureLab Ultra system from ELGA (High Wycombe, UK).

For aqueous mobile phases, 5 mM ammonium acetate, 0.1% formic acid and their mixture were separately tested and used along with the organic reagent, either methanol or acetonitrile.

Stock standard solutions of norethindrone, levonorgestrel, chlormadinone acetate and mifepristone were prepared in methanol at a final concentration of 1.0 mg/mL of each.

For measurements of linearity and limit of detection (LOD), blank blood samples in aliquot of 1.0 mL were firstly spiked with the standard mixture of target compounds at the concentrations of 0.1, 0.5, 1, 5, 10, 20, 50, 100, 200, 500 ng/mL, and then separately deproteinized by addition of acetonitrile at a volume ratio of 1:2, and finally centrifugated at 8000 r/min for 5 min. The resultant supernatant was received for further analysis.

For determination of extraction recovery, blank blood samples in aliquot of 1.0 mL were spiked with the standard mixture of target compounds at the concentrations of 5, 50, 500 ng/mL before or after deproteinization, respectively, and then centrifugated at 8000 r/min for 5 min to obtain the supernatant.

For matrix effect test, blank blood samples were deproteinized and centrifugated with the same procedures as above. The resulting supernatants or ultra-pure water were separately spiked with the standard at different concentrations of 5, 50 and 500 ng/mL.

Real samples were also deproteinized and centrifugated with the same procedures as above.

All samples were filtrated through the membranes of 0.22-μ m pores before LC-MS/MS analysis.

Analysis was performed on an Exion LC-QTRAP 5500 triple quadrupole system (AB Sciex, USA). Chromatographic separation was carried out by a Hypersil Gold column (50 mm × 2.1 mm, particle size 1.9 μ m, Thermo Fisher Scientific, USA) at room temperature. The injection volume was 2 μ L. The mobile phase consisted of two eluents: eluent A being methanol and B, the 5 mM ammonium acetate plus 0.1% formic acid in water. A gradient was run at a flow rate of 0.4 mL/min, starting with 10% A for 0.5 min and then increasing to 90% A within 2 min and being kept for 3 min before returning to initial condition in 0.5 min. An equilibration time of 2.5 min was allowed before the next injection.

Ionization was performed with an electrospray ionization (ESI) operation in positive ionization mode. The following parameters were chosen for all substances: spray voltage 5500 V; curtain gas= 35 psi; GS1= 55 psi; GS2= 55 psi; temperature= 550 ℃; dwell time = 50 ms. Other instrument parameters, e.g., declustering potential and collision energy were optimized by direct infusion of 20 ng/mL freshly-prepared standard solutions in methanol at a flow rate of 5 μ L/min. The precursor ion for each analyte was mass-selected by the first quadrupole and fragmented through a combination of the second quadrupole and collision energies to obtain the product ions. Analysis was conducted by multiple reaction monitoring (MRM) mode. Two product ions were used for qualitative analysis and one of them was used for quantification. The instrument control and data acquisition were guided by the operating software of Analyst 1.6.3. LC-MS/MS parameters for each analyte, including retention time (Rt), precursor ion (Q1), product ion (Q3), declustering potential (DP), collision energy (CE) were summarized in Table1.

Table 1

Table 1

Table 1 LC-MS/MS parameters for different drugs Analyte Rt /min Q1 /(m/z) Q3/ (m/z) DP /V CE/V norethindrone 2.49 299.1 109.1/231.2 50 37/28 levonorgestrel 2.57 313.2 245.2/295.2 35 27/23 chlormadinone acetate 2.61 405.1 309.2/345.1 80 23/19 mifepristone 2.62 430.2 372.3/288.1 35 32/47

Table 1 LC-MS/MS parameters for different drugs

LC separation was optimized with the analytical standards spiked in pure solvent. As elution gradient and solvent strength are main factors to affect the retention time, peak shape and resolution of analytes under certain flow rate ^[4], gradient elution, based on our previous research ^[5], was adopted to separate the target drugs and methanol or acetonitrile was individually tested as organic eluent when 0.1% formic acid was used as aqueous phase. The experimental data showed that signals of mifepristone had no obvious change with the usage of either eluent while norethindrone, levonorgestrel and chlormadinone acetate achieved their dramatically increasing sensitivities under methanol choice. Further study revealed that all of the analytes got strongest response when the mixture of 5 mM ammonium acetate-0.1% formic acid was applied as aqueous phase, compared with the performance from use of the single component that constituted the eluent. In order for the target substances to realize fast and sensitive analysis, a mobile phase of methanol/water combined with 5 mM ammonium acetate and 0.1% formic acid was selected into gradient elution mode for the following experiments.

For MS detection, the declustering potential for a precursor and the collision energy for effective fragmentations of the selected precursor ions were separately optimized for maximum sensitivity over the range of 0~200 V and 5~130 V. The typical LC-MS/MS chromatograms of all analytes were shown in Fig.1.

Fig.1

	Figure Option ViewDownloadNew Window
	Fig.1 LC-MS/MS chromatograms of progestogen-like drugs (a: MRM chromatogram of all targeted progestogen-like drugs; b: Extracted ion chromatograms of different drugs)

In order to validate the established method and reveal its features, quantification was investigated through determination of standard-spiking blood samples. Working equations of the tested drugs were constructed by peak area (y) against concentration (x) as listed in Table 2, with their linear correlation coefficients being all above 0.999. The LODs (S/N =3), obtained from the analysis of blood samples spiked with low concentrations of analyte standards, were between 0.1 and 1.0 ng/mL for different compounds.

Table 2

Table 2

Table 2 Working equations and detection limits of the analytes Analyte Quantification ion pair/ (m/z) Working equation Linear range /(ng/mL) Correlation coefficient Detection limit /(ng/mL) Producibility /% Intra-day Inter-day norethindrone 299.1/109.1 y=1.15× e4x+3.95× e4 5~500 0.999 9 1.0 4.0 5.8 levonorgestrel 313.2/245.2 y =6.81× e3x +3.97× e3 1~500 0.999 7 0.5 1.5 3.6 chlormadinone acetate 405.1/309.2 y =1.45× e4x +3.12× e3 0.5~500 0.999 7 0.1 2.1 6.9 mifepristone 430.2/372.3 y =1.15× e4x +4.09× e3 1~500 0.999 6 0.5 2.8 6.1

Table 2 Working equations and detection limits of the analytes

The strategy adopted in sample preparation was quite simple, and possibility of target loss may only exist in the precipitation procedure for deproteinization. Thus, extraction efficiency was estimated with analytes’ concentrations at three different levels by comparison of the peak areas obtained when the analytes were added before or after deproteinization. The results in Table 3 demonstrated the satisfactory extraction recoveries of 84.4 ~ 97.0 %.

Matrix effects were determined by the measurement of neat standard against the spiked supernatant at three concentration levels, being calculated as the percentage of peak area in the presence/absence of matrix. No obvious matrix effect was observed according to the data shown in Table 3.

Table 3

Table 3

Table 3 Extraction efficiency and matrix effects of the LC-MS/MS assay for each analyte at low (5), medium (50) and high (500) concentrations (ng/mL, n=6) Analyte Extraction efficiency (%) Matrix effect (%) 5 50 500 5 50 500 norethindrone 95.0 92.4 97.0 98.6 98.4 102.1 levonorgestrel 91.1 92.6 93.8 106.1 98.5 99.3 chlormadinone acetate 96.5 88.3 89.7 108.3 99.0 98.6 mifepristone 90.4 84.4 87.5 103.9 99.8 107.3

Table 3 Extraction efficiency and matrix effects of the LC-MS/MS assay for each analyte at low (5), medium (50) and high (500) concentrations (ng/mL, n=6)