Chemicals and standards
Methanol, acetonitrile, and deionized water were purchased from J. T. Baker (USA). All standards of MC-LA, MC-LF, MC-LR, MC-WR, MC-LW, MC-LY, MC-RR, and MC-YR (95% purity, Fig. 1) were obtained from BePure (China). The internal standard leucine enkephalin was purchased from Zhenzhun Biologicals, China. Stock standard solutions of microcystins were prepared in methanol in an amber glass container and stored at − 20°C in the dark for a maximum of 24 months. The 1 mg microcystins/L working solution from the stock solutions was prepared daily in methanol.
Eight MCs stock solutions were prepared by mixing MC standards with methanol. MC solutions were prepared in different concentrations by adding desired volumes of stock solution to methanol. The prepared standard solutions with a concentration of 1.0 mg L− 1 were stored and removed in the dark at − 20°C. The leucine-enkephalin internal standard solution (50 µg L− 1) was prepared in methanol/water (1:1, v/v) and stored at − 3°C.
Optimization of chromatographic conditions
Several gradients were investigated to optimize the peak resolution and sensitivity and to minimize the run time, including altering the flow rate, gradient, and the concentration of acetonitrile reached with the resulting gradient. The MC congeners were detected on a triple quadrupole mass spectrometer using multiple reaction monitoring with transitions optimized manually to achieve maximum sensitivity.
Solid phase extraction (SPE) column
The separation efficiency of online SPE columns depends on their retainability to microcystins. Microcystins are polar substances, and chromatographic columns with strong color fillers are optimal for the separation of macromolecular substances. Therefore, we evaluated the separation efficiency of XBridge® C8 and Oasis® HLB C18 by comparing the chromatographic resolution of eight microcystin congeners (20 ng L− 1). Compared with the commonly used Oasis® HLB Direct Connect HP (20 µm, 2.1 mm×30 mm), XBridge® C8 is used for reversed phase extraction, which is more suitable for nonpolar to medium-polarity target compounds. The online SPE column used in this study is XBridge® C8 Direct Connect HP (10 µm, 2.1mm ×30 mm) to retain the strong polar substances of eight MCs eluted in the void volume of the Oasis® HLB C18 column.
Chromatographic resolution (R) is used to characterize the degree of separation of two adjacent chromatographic peaks, which equals to the ratio of the difference between the retention times of adjacent chromatographic peaks (t1 and t2) and the average peak width of the two chromatographic peaks (w1 and w2) (Eq. 1).
Where t1 is the retention times of first peak, t2 is the retention times of second peak, w1 is the width of the first peak, w2 is the second peak.
When R < 1, the two peaks overlap; when R = 1, the resolution can reach 98%; and when R = 1.5, the resolution can reach 99.7%. Usually R = 1.5 is used as a sign that two adjacent components have been completely separated.
Water spiking with internal standard and filter membranes
An appropriate internal standard can balance the change in the signal response intensity of the analyte caused by matrix interference under certain conditions and reduce the interference of the loss of analyte during sample pretreatment[27, 28]. Leucine enkephalin is considered an internal standard for microcystin determination. Xu et al. showed that microcystins and leucine enkephalin can be separated well and their recovery rates are similar owing to their similar structure [29].
Pure water samples need to be filtered before analysis to protect the instruments and reduce matrix effects. However, MCs can be lost because hydrophilic filter membranes can absorb partial MCs through hydrogen bonds. Therefore, the recovery of three different membranes were evaluated in this study: polyethersulfone (PES), polytetrafluoroethylene (PTFE), and mixed cellulose ester (MCE) filters.
Mobile phases
The two components of the mobile phase were termed as “A + B” in this study. Six different mobile phases were evaluated: (1) Methanol + Water; (2) Acetonitrile + water; (3) Methanol with 0.1% (v/v) formic acid + water with 0.1% (v/v) formic acid; (4) Acetonitrile with 0.1% (v/v) formic acid + water with 0.1 % (v/v) formic acid; (5) Acetonitrile with 0.25% (v/v) formic acid + water with 0.1% (v/v) formic acid; (6) Acetonitrile with 0.5% (v/v) formic acid + water with 0.1% (v/v) formic acid. The separation efficiency was evaluated by comparing the peak intensity of eight microcystin congeners with the column maintained at 35°C.
Gradient elution procedures
Four gradient elution procedures were employed: (1) the water phase was held at 100% for 4.1 min, followed by a decrease to 0% over 2.9 min, washed for 4 min at 100% before the next injection; (2) the water phase was held at 98% for 4.6 min, followed by a decrease to 25% over 5.4 min, washed for 2 min at 98% before the next injection; (3) the water phase was held at 95% for 4.1 min, followed by a decrease to 60% over 1.9 min and followed by another decrease to 5% over 3 min, washed for 3 min at 95% before the next injection; (4) the water phase was held at 95% for 6 min, followed by a decrease to 5% over 3 min, washed for 2 min at 95% before the next injection.
Operating conditions of UPLC-MS/MS
The sample was introduced into the injection loop and transferred to the online SPE column for analyte preconcentration. The online aqueous mobile phase continued to flow after sample loading to ensure matrix and salt removal. The analyte was eluted by back-flushing the online SPE column by the UPLC mobile phase and separated by the chromatographic column prior to MS/MS detection. The mobile phase of the binary pump was applied as described in Sect. 2.2.3.
Analysis of eight MCs was performed using an ACQUITY UPLC system coupled to a Xevo TQ-MS (triple quadrupole MS/MS) mass spectrometer (Waters, Manchester, UK). The analytical column and solid phase extraction column were ACQUITY UPLC® BEH C18 (1.6 µm, 2.1 mm × 50 mm) and XBridge®C8 Direct Connect HP (10 µm, 2.1 mm × 30 mm), respectively. The system was operated in the electrospray positive mode (ESI+), with the capillary voltage of 3.70 kV, source and desolvation temperatures of 150 and 500°C, respectively, desolvation gas flow of 1000 L h− 1, cone hole backflush gas pressure of 30 V, cone hole backflush gas flow rate of 50 L h− 1, and collision gas flow of 0.06 mL min− 1, optimized to produce the best sensitivity across all analytes. Detection and quantification were achieved using targeted analysis via positive ion scanning and multiple reaction monitoring. Other mass spectrometer parameters are presented in Table 1.
The flow rate was set at 0.4 mL min− 1 with the organic phase held at 2% for 8.1 min, followed by an increase to 75% over 2.1 min, washed for 0.8 min, and returned to 2%. The mobile phases of the quaternary pump included pure water (mobile phase A) and acetonitrile (mobile phase C). The flow rate was kept at 2.0 mL min− 1 with the water phase held at 100% for 4.1 min, followed by a decrease to 0 over 2.9 min, washed for 4 min at 100% before the next injection. The injection volume was set at 5 mL.
Table 1
Compound-dependent MS/MS parameters of eight MC congeners.
Compound
|
Precursor ion (m/z)
|
Quantification fragment (m/z)
|
Confirmation fragment (m/z)
|
Cone energy (V)
|
Collision energy (eV)
|
MC-LR
|
995.4
|
135
|
213
|
85
|
75
|
MC-YR
|
1045.6
|
135
|
213
|
85
|
85
|
MC-RR
|
519.7
|
135
|
440.4
|
42
|
32
|
MC-WR
|
1068.5
|
135
|
213
|
60
|
95
|
MC-LA
|
910.5
|
135
|
776
|
40
|
80
|
MC-LF
|
986.3
|
135
|
213
|
42
|
68
|
MC-LY
|
1002.6
|
135
|
985
|
45
|
80
|
MC-LW
|
1025.53
|
135
|
213
|
45
|
65
|
* Quantitative ion |
Method validation
To validate the method, a seven-point matrix of standards ranging from 0.01 to 1.00 ng mL− 1 was configured to match the calibration curve. The microcystin standard stock solution was serially diluted step by step to 0.01, 0.02, 0.05, 0.10, 0.20, 0.50, and 1.00 ng mL− 1. Sequential determination was performed under chromatographic analysis conditions. Linear regression was applied with the injection concentration (x) corresponding to the corresponding peak area (y). The internal standard of leucine enkephalin was added to 0.01 ng mL− 1, and the authenticity and absolute recovery of the analyte were calculated.
Low-, medium-, and high-concentration standard samples were measured six times in parallel according to the above method, and the average recovery rate and relative standard deviation (RSD) were calculated. The limit of detection (LOD, ng g− 1) and limit of quantification (LOQ, ng g− 1) were calculated using the signal-to-noise ratios of 3 and 10 based on the lower-end calibration curve levels. These values were calculated for 2 mL injection, which is equivalent to 2 pg injected on the column.
Six samples were combined into one batch; three batches of standards were added and recovery experiments were conducted using purified water; blank samples were added for quality control. The specific operation method described in Sect. 2.5 sets three concentrations of 0.05, 0.10, and 0.50 ng mL− 1 to test and calculate the recovery rate. The precision and recovery results are presented in Table 3.
Water samples for method development
We used pure water to optimize the chromatographic conditions (herein referred to as water MO), and 12 water samples (CH1, CH2, …CH12) collected from Chaohu Lake (Anhui Province, China) in August 2020 were used to validate the method. We chosed at least 12 zones in Chaohu Lake, used a water harvester to remove the surface scum, and collected ~ 500 mL water sample in bottles from each zone from the 0–50 cm depth. Next, ~ 100 mL of each water sample was in-situ filtered using a 0.45 µm cellulose acetate filter membrane (JiuDing, China) to a 120 mL polypropylene bottle. The samples were then placed in a cooler with ice packs and transferred to the laboratory for further treatment. Twelve sampling points are shown in Fig. 1.
After 20 mL of water MO was filtered using a disposable medical syringe coupled with a 0.22 µm filter in the injection vials, 10 ng L− 1 internal standard was spiked to each sample prior to injection. To quantify MCs, a seven-point mixed standards calibration curve in the range of 0.01– 1.00 ng mL− 1 was created based on the initial sample size of 20 mL.