2023 Volume 48 Issue 4 Pages 137-148
A method of quantifying glyphosate (Gly) in human urine by means of MonoSpin TiO extraction and 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) derivatization with isotope dilution mass spectrometry (IDMS) was investigated and optimized. The method’s quantification limit under optimized conditions was 0.3 µg/kg for FMOC-Gly, which was comparable to or lower than those described in previous studies. When a spike test using human urine samples was carried out with optimized analytical conditions, the trueness for FMOC-Gly was as follows: 101.6–104.9% for a spike level of 0.5 µg/kg and 99.2–101.0% for a spike level of 30 µg/kg. The intra-day repeatability and inter-day reproducibility were <6.5%. The spike test results for validation between the “with” and “without” derivatization methods were comparable at 1 µg/kg. Our results indicate that using MonoSpin TiO extraction and FMOC-Cl derivatization with IDMS is an accurate method for analyzing Gly in human urine.
Glyphosate (N-(phosphonomethyl)glycine; Gly) is categorized as a phosphonic and amino acid herbicide1) and is one of the most widely used classes of pesticides in the world.2,3) The current annual use of Gly worldwide is estimated at about 600,000–750,000 tons and is expected to reach 740,000–920,000 tons by 2025.4,5) Gly has been detected in some foods2,6) and environmental samples.3,7) The safety of Gly is still being discussed.8) The International Agency for Research on Cancer (IARC) monographs9) describe Gly as “probably carcinogenic to humans (Group 2A)” although the European Food Safety Authority (EFSA) identified no apparent risk to consumers.8,10) Furthermore, exposure to Gly may adversely affect reproductive health such as shortening the length of gestation,11) and may negatively affect the mitochondria respiration efficiency of human sperm.12) Therefore, Gly contained in human biological samples such as urine has been analyzed,13,14) e.g., to assess the relationship between the level of exposure to Gly and its health effects, which requires accurate analytical results.
The objectives of the present study were to optimize and establish an accurate method for the analysis of Gly in human urine. The main metabolite of Gly found in the environment and plants is aminomethylphosphonic acid (AMPA)15,16); however, it was not selected as a target analyte because the results of human experimental studies suggest that Gly is excreted in urine in a mostly unchanged form,8,17) and AMPA is not considered to be of greater toxicological concern than its parent compound.18) In addition, glufosinate (Glu), mainly used to control Gly-resistant weeds,5) was not selected because the detection rates were considerably low.5,15,19) Accurate analysis of Gly at low concentration levels such as in urine samples is challenging due to its high polarity, poor detectability, and low molecular weight.5,6,20,21) Various methods are used to analyze Gly in human urine. Currently, one of the simplest measurement methods is to measure Gly directly by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) with anion exchange or hydrophobic interaction liquid chromatography (HILIC) columns22) without derivatization. Thus, we initially examined the “without derivatization” method. On the other hand, in case of insufficient sensitivity of this method for quantifying trace amounts of Gly in human urine (e.g., the method quantification limit (MQL) of >1.0 µg/kg), we also examined derivatization with 9-fluorenylmethoxycarbonyl chloride (also known as 9-fluorenylmethyl chloroformate, FMOC-Cl). To analyze contaminants in biological samples such as urine, complex sample pretreatment (extraction and cleanup) as well as highly selective instrumental analyses are needed. Preferably, a pretreatment method should be developed that is not only accurate but also simple for the analysis of many samples such as in an epidemiological study. In many cases, solid phase extraction (SPE) such as anion and/or cation exchange and zirconia-coated silica are used for the pretreatment process of Gly in biological samples.5,23–25) Examples of previous studies on analytical methods are listed in Table 1.5,15,22–27) The present study examined the MonoSpin TiO extraction method for the analysis of Gly at low concentration levels in human urine. MonoSpin TiO is a monolithic SPE column coated with titanium dioxide (also known as titania28)), and the method using MonoSpin TiO can be easily and quickly implemented by centrifuge29,30) compared with conventional SPE methods. In addition, isotope dilution mass spectrometry (IDMS), which has the potential to be a primary measurement method,31–34) was applied to ensure a reliable analysis. Compared with external or internal standard methods, IDMS generally delivers higher accuracy and precision35) if the analysis (including extraction, cleanup, and instrumental measurement) is carried out under adequate conditions. Our optimized established method was validated by a spike test and a comparison of the results of the two methods, i.e., “with” and “without” derivatization.
| Target analytes | Internal standards | Derivatization reagent | Sample amount | SPE used for cleanup process | Instrumental analysis | Separation LC column | LODs and LOQsb) | Reference |
|---|---|---|---|---|---|---|---|---|
| Gly, AMPA, Glu, MPPA, BIAL | DL-2-amino-3-phosphonopropionic acid | TMOA and acetic acid | 10 µL (for both serum and urine sample)a) | N/A (Deproteinization for serum and no pretreatment for urine) | LC-MS (ESI+) | Symmetry C8 (Waters, MA, USA) | LODs for serum: 50 µg/L (Gly and MPPA), 30 µg/L (Glu), 100 µg/L (BIAL), 200 µg/L (AMPA); LODs for urine: 50 µg/L (Gly, Glu, and MPPA), 400 µg/L (BIAL), 200 µg/L (AMPA) | 26)a),b) |
| Gly, Glu, BIAL | Gly-13C2,15N | TMOA and acetic acid | 100 µL (for both serum and urine sample)a) | HybridSPE-Phospholipid (Merck; Zirconia-coated silica) cartridge | LC-MS/MS (ESI+) | Acquity UPLC BEH C18 (Waters) | LODs for serum: 50 µg/L (Gly and Glu), 200 µg/L (BIAL); LOQs for serum: 200 µg/L (Gly), 100 µg/L (Glu), 500 µg/L (BIAL); LODs for urine: 100 µg/L (Gly and Glu), 400 µg/L (BIAL); LOQs for urine: 400 µg/L (Gly), 200 µg/L (Glu), 1000 µg/L (BIAL) | 23)a),b) |
| Gly, AMPA, Glu | Gly-13C,15N; AMPA-13C,15N, D2; Glu-D3 | FMOC-Cl was the best method in this study (compared to underivatized or ImS-Cl) | 0.5 mL | Strata-X (Phenomenex, Torrance, CA, USA) and SepPak C18 (Waters) (usable by target compounds) | LC-MS (ESI(+)-HRAM-MS) | Kinetex-C18-Evo (Phenomenex) | LOD: 0.037 µg/L (Gly), 0.020 µg/L (AMPA), and 0.007 µg/L (Glu) for the best method of each compound | 24)b) |
| Gly | Gly-13C2,15N | (N/A) | 1 mL diluted sample (Diluted with H2O to a creatinine concentration of 0.05 g/L) | Creatinine-matching dilution method/ISOLUTE-96 SCX and NH2 (Biotage, Uppsala, Sweden) as SPE | LC-MS/MS (ESI+) | Scherzo SM-C18 MF (Imtakt, Kyoto, Japan) | LOD: 0.1 µg/L; LOQ: 0.3 µg/L (Gly) | 25) |
| Gly, AMPA, Glu, MPPA | Gly-13C,15N; AMPA-13C,15N, D2; Glu-D3 hydrochloride; MPPA-D3 | 2,3,4,5,6-pentafluorobenzyl bromide | 100 µL | N/A (The flash freeze technique was used to speed up the extraction process and require less organic solvent.) | LC-MS/MS (ESI+) | HSS T3 for MPPA and BEH Phenyl for other analytes (Both columns were from Waters) | LODs: 0.095 µg/L (MPPA), 0.086 µg/L (AMPA), 0.077 µg/L (Gly), 0.084 µg/L (Glu); LOQs: 0.32 µg/L (MPPA), 0.29 µg/L (AMPA), 0.26 µg/L (Gly), 0.28 µg/L (Glu) | 15) |
| Gly | Gly-13C2,15N | FMOC-Cl | 1000 µL | N/A (Hydrochloric acid (37%) and methanol were added to the urine sample, and this was shaken and centrifuged.) | LC-MS/MS (ESI+) | Acquity UPLC BEH C18 (Waters) | LOQ: 0.50 µg/L (Gly) | 22) |
| Gly and AMPA | (N/A) | FMOC-Cl | 1 mL for lyophilized sample and 5 mL for without lyophilization | N/A (The urine samples were lyophilized for 24 hr.) | LC-MS/MS (ESI+) | Acquity UPLC BEH C18 (Waters) | LODs: 0.5 µg/L (Gly) and 0.1 µg/L (AMPA); LOQs: 1 µg/L (Gly) and 0.5 µg/L (AMPA) | 27) |
| Gly, AMPA, Glu | Gly-13C2,15N; AMPA-13C,15N, D2; Glu-D3 | (N/A) | 250 µL | Oasis MAX (anion exchange) and MCX (cation exchange) cartridges (Waters) | LC-MS/MS (ESI-) | Gemini C6-Phenyl column (Phenomenex) connected to a Betasil C18 guard column (Thermo Fisher Scientific, MA, USA) | MDLs: 0.14 µg/L (Gly), 0.12 µg/L (AMPA and Glu); MQLs: 0.48 µg/L (Gly), 0.40 µg/L (AMPA), 0.41 µg/L (Glu) | 5)b) |
| Gly | Gly-13C3,15N | (N/A) | 0.2 g | MonoSpin TiO column (GL Sciences) | LC-MS/MS (ESI+) | HILICpak VT-50 2D (Shodex) | MDLs: 0.3 µg/kg; MQLs: 1.0 µg/kg | This studyc) |
| Gly | Gly-13C3,15N | FMOC-Cl | 0.2 g | MonoSpin TiO column (GL Sciences) | LC-MS/MS (ESI+) | InfinityLab Poroshell 120 CS-C18 (Agilent Technologies) | MDLs: 0.1 µg/kg; MQLs: 0.3 µg/kg | This studyc) |
a) Human serum was also the target matrix. b) The expression for the unit of each original paper is arranged by the author of this paper as needed. c) The unit is expressed as µg/kg because IDMS was used. Gly: glyphosate; Glu: glufosinate; AMPA: aminomethylphosphonic acid; MPPA: 3-[hydroxy(methyl)phosphinoyl]propionic acid; BIAL: bialaphos; FMOC-Cl: 9-fluorenylmethoxycarbonyl chloride; TMOA: trimethyl orthoacetate; ImS-Cl: 1-methylimidazole-sulfonyl chloride; SPE: solid phase extraction; LOD: limit of detection; LOQ: limit of quantitation; MDL: method detection limit; MQL: method quantification limit; HRAM: high-resolution accurate-mass; N/A: not applicable
Two types of pooled human urine (samples A and B) were prepared by BioIVT (Westbury, NY, USA) and obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Each pooled sample was obtained from two healthy donors and 0.2 µm filtered. The samples were stored at a temperature of about −80 °C until analysis, at which point they were thawed at room temperature and sufficiently mixed for homogenization. The target compound (Gly) was fortified to ensure the appropriate concentration levels (details are provided later).
2. Chemicals and reagentsAcetonitrile (ACN) was purchased from Kanto Chemical (Tokyo, Japan) for pesticide residue and polychlorinated biphenyls analysis grade and from FUJIFILM Wako Pure Chemical for LC/MS grade. Ammonia solution, sodium tetraborate (anhydrous), acetic acid (reagent grade), and methanol (pesticide residue and polychlorinated biphenyls analysis grade) were purchased from Kanto Chemical. Trifluoroacetic acid (TFA, Wako special grade), ammonium acetate, phosphoric acid (reagent grade), formic acid (LC/MS grade), and FMOC-Cl (peptide synthesis grade) were purchased from FUJIFILM Wako Pure Chemical. Purified water (Puric α, UP-0090α-TU1, Organo Corp., Tokyo, Japan) was used for preparing the solutions for the internal standard, syringe spike (used for calculation of the recoveries), calibration, TFA, ammonia, sodium tetraborate, phosphoric acid, formic acid, and ammonium acetate.
3. Preparation of internal standard and syringe spike solutionsTo reduce the adsorption of Gly on glass,25,36) all solutions were prepared by using polypropylene bottles and pipette tips. Internal standard solutions were gravimetrically prepared by dissolving isotope labeled pesticides: Gly-13C3,15N (Cambridge Isotope Laboratories, Cambridge, MA) in water. The syringe spike solution was also gravimetrically prepared by dissolving acetamiprid-13C6 (Cambridge Isotope Laboratories) in water.
4. Analytical methodThe analysis was separated into two parts as shown in the flowchart in Fig. 1. Part 1 is based on the documentation of GL Sciences37) and Shodex.38) Part 2 is based on the documentation of Agilent Technologies.39)
The glassware was not used in all the analytical processes, similarly to the preparation of solutions as described above. The human urine sample (0.2 g), weighed in a 1.5 mL microcentrifuge tube, was spiked with the internal standard solution and then dried by a nitrogen gas stream on a heat block at 110 °C. Solution B (50/50 (v/v) ACN/water solvent with 0.1% TFA) in the amount of 400 µL was added to the dried sample, and the sample solution was loaded on a MonoSpin TiO column (GL Sciences, Tokyo, Japan) after being conditioned with 50 µL of solution A (80/20 (v/v) ACN/water solvent with 0.1% TFA) and 50 µL of solution B at 3000 rpm for 1 min by a compact centrifuge (MF-12000, AS ONE, Osaka, Japan). This operation, except for the column conditioning process, was repeated twice. The sample of 800 µL (400 µL ×2) solution containing Gly was centrifuged at 3000 rpm for 5 min. After reloading the sample solution on the MonoSpin TiO column, the solution was centrifuged again at 3000 rpm for 5 min and the collected solution was discarded. The MonoSpin TiO column was washed with 50 µL of solution B and 50 µL of solution A (centrifuged at 3000 rpm for 1 min), and the collected solution was discarded. Gly was eluted with 300 µL of 5% ammonia solution (centrifuged at 3000 rpm for 5 min) followed by drying using a nitrogen gas stream on the heat block at 110 °C. For the condition study of MonoSpin TiO, the syringe spike solution (0.15 g) was added to the cleaned-up sample. Gly was analyzed by LC-MS/MS (LCMS-8030 quadrupole mass spectrometer, Shimadzu, Kyoto, Japan and a LC-20A series HPLC system, Shimadzu, equipped with a HILICpak VT-50 2D column (2.0×150 mm, 5 µm, Shodex, Tokyo, Japan)). Based on a previous study,40) a polyether ether ketone (PEEK) tube was used instead of metal tubing wherever possible and the LC flow path (separation column was removed) was coated with a 2% phosphoric acid solution for 12 hr at 0.05 mL/min to avoid interaction between the Gly and the metals.25) The mobile phase was a water/aqueous solution of 1% formic acid/ACN (70/20/10, v/v/v37)) in the isocratic elution at a flow rate of 0.3 mL/min. The column temperature was set at 40 °C and the injection volume was 50 µL. The target analytes were ionized by electrospray ionization (ESI) in the positive mode. The LCMS-8030 parameters were a desolvation line (DL) temperature of 250 °C, heat block temperature of 400 °C, nebulizer gas flow of 1.5 L/min, and drying gas flow of 10.0 L/min. The m/z of the ions for quantification and the results of multiple reaction monitoring (MRM) optimization (collision energy, and Q1 and Q3 prerod bias) are shown in Table 2.
| Compound | Transition (quantification) | Q1 Pre Bias (V) | CE | Q3 Pre Bias (V) |
|---|---|---|---|---|
| Gly | 169.70>88.10 | −12 | −10 | −15 |
| Gly-13C3,15N | 173.70>92.05 | −12 | −10 | −15 |
| FMOC-Gly | 392.00>88.05 | −27 | −21 | −15 |
| FMOC-Gly-13C3,15N | 395.90>92.05 | −27 | −22 | −15 |
| Acetamiprid-13C6 (Syringe spike) | 229.10>56.10 | −15 | −17 | −20 |
MRM: multiple reaction monitoring, Pre: prerod
The spike test was performed on human urine sample A for optimization of the MonoSpin TiO conditions (spike level: 30 µg/kg). Examined processes were as follows (the exam number corresponding to each study is also shown in Fig. 1, n=3): type of solution for MonoSpin TiO (Exam 1-1), volume of additive for solution B (Exam 1-2; 500 µL and 800 µL), TFA concentration in solutions A and B (Exam 1-3; 0.05%, 0.10%, and 0.50%), volume of washing solution (Exam 1-4; 20 µL, 50 µL, and 200 µL), volume of eluate by ammonia solution (Exam 1-5; 50 µL, 300 µL, and 500 µL), concentration of ammonia solution for eluate (Exam 1-6; 2.0% and 5.0%), speed of centrifugation (Exam 1-7; 3000 rpm and 5000 rpm), and time of centrifugation after sample loading (Exam 1-8; 5 min and 10 min). The basic conditions were as follows: volume of solution B=800 µL, TFA concentration=0.10%, volume of washing solution=50 µL, volume of ammonia solution=300 µL, concentration of ammonia solution=5.0%, centrifugation speed=3000 rpm, and centrifugation time=10 min. The examinations were carried out by changing each condition, and the conditions that resulted in the highest recoveries in each examination were adopted as the optimized conditions. The chromatographic separation of Gly with optimized conditions at a spike level of 1 µg/kg was also observed to confirm the cleanup efficiency.
7. Preparation of calibration solutions for method part 1The calibration solutions were prepared as follows: Gly solutions were gravimetrically prepared by dissolving the Gly standard (TraceSure grade, FUJIFILM Wako Pure Chemical) in water. The solution was mixed gravimetrically with the internal standard (Gly-13C3,15N) and syringe spike solutions as prepared above, and was used for calibration. Moreover, by mixing the final calibration solution with cleaned-up extracts of blank urine, matrix-matched calibration solutions were prepared. Due to the good linearity, as described below, the one-point calibration method was applied. The solution was prepared in such a way as to match as closely as possible to the final concentration of Gly in the cleaned-up extracts of the human urine samples.
8. Method part 2 (derivatization by FMOC-Cl)Part 2 was continued from part 1, i.e., the cleaned-up dried sample before adding the syringe spike solution of part 1 was used for part 2 as the beginning of the analysis. Exactly 70 µL of water, 10 µL of 5.0% sodium tetraborate buffer solution, and 10 µL of 0.1% FMOC-Cl solution in ACN (freshly prepared just before the reaction22)) were added to the samples in the microcentrifuge tubes, which were incubated on the heat block for 20 min at 50 °C. To stop the derivatization reaction, 10 µL of 2% phosphoric acid solution was added to the microcentrifuge tubes. Then, the syringe spike solution (0.05 g) was added to the samples, and the samples were measured using LC-MS/MS (same model as for part 1). LC-MS/MS was operated as follows: separation column: InfinityLab Poroshell 120 CS-C18 (2.1×150 mm, 2.7 µm, Agilent Technologies, Santa Clara, CA, USA) with a guard column; InfinityLab Poroshell 120 CS-C18, 2.1×5 mm, 2.7 µm, Agilent Technologies); mobile phases A and B consisted of a 5 mM aqueous solution of ammonium acetate and ACN, respectively; gradient program: B was increased from 5% to 65% in 14 min and from 65% to 95% in 5.5 min, and was then decreased from 95% to 5% in 2.5 min; flow rate: 0.15 mL/min; column temperature: 40 °C; injection volume: 50 µL; ionization: ESI in the positive mode. LCMS-8030 parameters were as follows: DL temperature of 250 °C, heat block temperature of 400 °C, nebulizer gas flow of 3 L/min, and drying gas flow of 15 L/min. The m/z of the ions for quantification and the results of multiple reaction monitoring (MRM) optimization (collision energy, and Q1 and Q3 prerod bias) are shown in Table 2.
9. Optimization study for FMOC-Cl derivatization conditions in method part 2Optimization of the FMOC-Cl derivatization conditions was performed by spiking Gly using human urine sample A (spike level: 30 µg/kg). Examined processes were as follows (exam number corresponding to each study is also shown in Fig. 1, n=3): concentration of sodium tetraborate buffer solution (Exam 2-1; 2.5% and 5.0%), volume of sodium tetraborate buffer solution (Exam 2-2; 10 µL, 30 µL, and 60 µL), FMOC-Cl concentration (Exam 2-3; 0.05%, 0.10%, and 0.50%), reaction temperature (Exam 2-4; 25 °C, 50 °C, and 80 °C), and reaction time (Exam 2-5; 20 min and 40 min). The basic conditions were as follows: concentration of sodium tetraborate buffer solution: 5.0%, volume of sodium tetraborate buffer solution: 10 µL, FMOC-Cl concentration: 0.10%, reaction temperature: 50 °C, and reaction time: 20 min. The examinations were carried out by changing each condition. To accurately evaluate the conditions, in addition to comparing the area in the chromatogram of FMOC-Gly obtained by each condition, the obtained results were also corrected using the area and the addition amount of the syringe spike by Eq. (1).
 | (1) |
The conditions that resulted in the highest reaction efficiencies in each examination were adopted as the optimized conditions.
10. Preparation of calibration solutions for the overall analytical method (part 1+2)The calibration solutions were prepared as follows: Gly solutions were gravimetrically prepared by dissolving the Gly standard in water and this solution was mixed gravimetrically with the internal standard (Gly-13C3,15N) as prepared above. This calibration was derivatized by FMOC-Cl according to part 2, and the obtained calibration with derivatization was used for the overall analytical method (part 1+2) after adding the syringe spike solution. Furthermore, the matrix-matched calibration solutions were prepared as follows: the solution (mixture of Gly and Gly-13C3,15N) was mixed with blank urine (cleaned-up dried extracts obtained by part 1), and this mixture was derivatized by FMOC-Cl according to part 2 as described above.
11. Evaluation of accuracy (trueness and precision) for the overall analytical method (part 1+2)The overall analytical method (part 1+2) was evaluated by a spike test for Gly in human urine sample A (two spike levels: 0.5 µg/kg (based on the lower limit of quantitation by a previous study using FMOC-Cl derivatization22) and set as near the MQL) and 30 µg/kg (set as high concentration)). The obtained results were evaluated as percentages by the quantification results of IDMS relative to the spiked amount of Gly. The chromatographic separation of FMOC-Gly was also observed.
12. Spike test using another sample of human urine (sample B)The spike test for optimizing the analytical method was also carried out by using sample B to determine whether it is possible to analyze Gly in another human urine sample. The results of the spike test were evaluated by three replicates on Day 1, Day 2 and Day 3 using the same instrument and operator, and the relative standard deviations (RSDs) were calculated for assessing the intra-day repeatability (RSDr, n=3) and the inter-day reproducibility (RSDR, n=9). The analysis (part 1+2) was performed as described above.
13. Comparison of accuracy between method part 1 and 1+2 (sample B)The spike test for the optimized method part 1 and part 1+2 was carried out by using sample B at 1 µg/kg to validate an established method. The analysis was performed as described above. The obtained results were evaluated as percentages by the quantification results of IDMS relative to the spiked amount of Gly and were compared.
To determine the linearity of the response, five calibration solutions were injected for LC-MS/MS. In the range covering the final concentration of Gly in the cleaned-up extracts (0.3–50 µg/kg), the correlation coefficient was satisfactory (r >0.9998 for Gly in method part 1, and r >0.9995 for FMOC-Gly in method part 1+2). The precision, represented as RSDs, at 1.0 µg/kg was 1.5% for part 1 and 1.2% for part 1+2 (n=3), which was also satisfactory for an accurate analysis.
2. Optimization study for MonoSpin TiO conditions of method part 1MonoSpin TiO is a monolithic SPE column coated with titanium dioxide (also known as titania28)) and tends to specifically bind to phosphate groups.30) It is suggested that the separation mechanism is due to the Lewis acid–Lewis base interaction between titania and the phosphate group.41) Although Saito et al.30) used MonoSpin TiO for the analysis of Gly (and Glu) in human serum as an accurate analytical method, the recoveries were very low (2.1–2.3% for Gly). We performed the analysis according to this method using 0.1% acetic acid, methanol, water, ACN, and ACN-ammonia (9 : 1) solutions for MonoSpin TiO (Exam No. 1-1 in Fig. 1). However, Gly could not be recovered (<method detection limit (MDL)) from the urine samples. Therefore, optimization of the MonoSpin TiO conditions was examined by using other solutions, that is, solution A and B, and an ammonia solution as described above, which was based on the study by Ohta et al.37) Ohta et al.37) also evaluated a monolithic silica spin column coated with titanium dioxide for the analysis of Gly (and Glu, AMPA, 3-[hydroxy(methyl)phosphinoyl]propionic acid (MPPA), and bialaphos) in several types of samples, and the recovery for the urine sample was 67.2% for Gly (concentration: 10 ppm). The recoveries were good; however, it is necessary to optimize the analytical conditions in order to apply the method to a general low concentration level in urine. For example, the concentrations of Gly detected in urine samples from the populations of Iowa (n=10) and New York (n=10) of the Unites States were 1.18 ng/mL (mean value calculated from the concentrations of more than the MQL, in six out of ten samples) and 0.53 ng/mL (detected in only one sample), respectively,5) and the concentrations for Japanese people (children, adults, and farmers) were 0.25 µg/L as the 50th percentile and 1.99 µg/L as the maximum.25) Therefore, the conditions for MonoSpin TiO extraction were optimized in the present study.
The optimization results for each parameter (Exam No. 1-2 to 1-8 shown in Fig. 1) are given in Fig. 2. The volume and concentration of the TFA solution are considered to be important factors for the retentivity and selectivity of phosphorus compounds. For Exam No. 1-2 (volume for solution B) and 1-3 (TFA concentration in solutions A and B), our results showed that 800 µL (maximum volume available for the microcentrifuge tube used in our method) and 0.10% (pH=3) were higher recoveries compared to the other conditions in Exam 1-2 and 1-3, respectively. These results indicated that the conditions were balanced with retentivity and selectivity for Gly in human urine. A previous study42) indicated that the amount of adsorption of phosphate ion to TiO2·H2O was the maximum at pH=3, which is consistent with our result. For the washing solution (Exam No. 1-4), we selected 50 µL as an appropriate volume because a volume of >50 µL was only a slightly higher recovery for Gly than that of 20 µL. The optimized volume and concentration for the eluate (ammonia solution) were 300 µL and 5.0% in Exam No. 1-5 and 1-6, respectively, since the recoveries were significantly higher compared to those of the other conditions. The volume of 50 µL was a low amount just for the elution of Gly, and the lower recoveries obtained by 500 µL are considered to be due to interferences in the LC separation of isotope-labeled Gly by coexisting matrix compounds probably eluted by too much eluate (500 µL) although the interfering compounds could not be identified. For the concentration, the target compounds were not sufficiently collected by 2.0%, as shown in Fig. 2, possibly because of a low elution efficiency. The optimized centrifugation speed and time were 3000 rpm and 5 min in Exam No. 1-7 and 1-8, respectively. Depending on the volume and type of sample, a certain level of slow centrifugation speed tends to increase the recoveries,28) and our results were consistent with this tendency. For the centrifugation time, we selected 5 min because there was no significant difference in the recoveries for 5 and 10 min. The highest recovery obtained by the analysis with optimized conditions for Exam No. 1-1 to 1-8 was 81.2±11.2% (n=3, mean±standard deviation (SD)), and these results satisfied the accuracy requirement (in the range of 70–120%43,44)). Obtained chromatograms of Gly using the optimized conditions for a spike level of 1.0 µg/kg are shown in Fig. 3, and there were no interferences in the LC separation for Gly and Gly-13C3,15N. This result indicates that MonoSpin TiO with optimized conditions could sufficiently clean up the human urine sample for the analysis of Gly.
To determine the instrumental detection limit (IDL) and instrumental quantification limit (IQL), the standard solutions were injected into the LC-MS/MS. The IDL was defined as the absolute amount of analyte that gave a signal-to-noise ratio (S/N) of 3, and the IQL was defined as the absolute amount of analyte that gave a S/N of 10 (Table 3). The MDL and MQL for part 1 are also listed in Table 3. The MDL was defined as the concentration detected by part 1 that gave a S/N of 3, and the MQL was similarly defined as a S/N of 10. The MQL obtained by the optimized method part 1 was 1.0 µg/kg, which seemed insufficient for the analysis of Gly in human urine based on previous studies although this result was not considered to be poor. Thus, the FMOC-Cl derivatization method was also examined.
| Compound | IQL (pg) | IDL (pg) | MQL (µg/kg) | MDL (µg/kg) |
|---|---|---|---|---|
| Gly | 15 | 5.0 | 1.0 | 0.3 |
| FMOC-Gly | 3.8 | 1.3 | 0.3 | 0.1 |
To enhance the detection sensitivity, derivatization with FMOC-Cl, which quickly reacts with primary and secondary amines,24,45) is one of the most frequently used methods for the analysis of Gly.22,24,46) In the present study, the optimization of FMOC-Cl derivatization conditions was examined for analyzing lower concentrations than the MQL of method part 1. To the best of our knowledge, no studies have applied FMOC-Cl derivatization combined with MonoSpin TiO (method part 1) and IDMS for the analysis of Gly in human urine samples.
The results for the optimization of each parameter (Exam No. 2-1 to 2-5 shown in Fig. 1) are given in Fig. 4. The most widely used buffer for the derivatization reaction is borate, the pH of which is an important factor in this experiment.22,47) In previous studies, pH of 8.0–10.0 was often adopted as an appropriate condition.22,47) For Exam No. 2-1 (concentration of sodium tetraborate buffer solution) and Exam No. 2-2 (volume of sodium tetraborate buffer solution), our results showed that 5.0% and 10 µL were suitable conditions, respectively. In previous studies,44,48) 2.5% and 5.0% were examined as borate concentrations (5.0% solution was prepared by heating at 50 °C until complete dissolution44)). Although there were no significant differences between the two concentrations, 5.0% (pH=9) was selected as an optimal condition because the repeatability was better than that for 2.5%. An unsuitable repeatability for 2.5% may be caused by insufficient buffering capacity with a lower concentration to complete the derivatization. Our optimized conditions were consistent with those of a previous study, which indicated that a pH of 9 is optimal for the derivatization.49–51) Different volumes of 5.0% borate buffer solution (10 µL, 30 µL, and 60 µL) were used in a volume study, and the reaction efficiency for 60 µL was slightly higher than that for 10 µL and 30 µL (provided that the differences between them were not large). On the other hand, it is also possible that the presence of more borate in the samples could cause peak tailing.47) Thus, we selected 10 µL as an appropriate volume. The optimized FMOC-Cl concentration was 0.10% in Exam No. 2-3 because the reaction efficiencies for FMOC-Gly were the highest compared to those of 0.05% and 0.50%, as shown in Fig. 4. The concentration of 0.10% for FMOC-Cl is consistent with the conditions in previous studies.39,48) Our result indicated that 0.05% of FMOC-Cl was a low concentration for achieving complete derivatization. A higher concentration such as 0.50% increases the production of FMOC-OH, which is the by-product formed by the reaction between FMOC-Cl and water.22,46,50) FMOC-OH can interfere with analyte detection because of its poor solubility in water.22,46,50) In addition, FMOC-OH can precipitate, which would disturb the performance of the chromatographic column and reduce the efficiency of the ionization process in the MS source.22,46,50) In the present study, after carefully examining the derivatization conditions, a guard column was additionally used to prevent even a small amount of FMOC-OH from precipitating to the separation column. To facilitate the reaction of the derivatization, it is also necessary to determine the ratio of ACN (that is, the FMOC solution in ACN) in preparing the derivatized sample.46) In previous studies, the chromatographic signals were better in the presence of 10% ACN.47,50) It is also reported that there were no changes in the derivatization yields between the range of 10% and 65% for ACN.46) Based on these studies, 10 µL of 0.1% FMOC-Cl solution in ACN was added to the microcentrifuge tube containing 70 µL of water and 10 µL of buffer solution, as shown in Fig. 1. Because the final solution volume related to MDL and MQL was taken into consideration, the presence of ACN was slightly more than 10%. However, there were no problems with the chromatogram and sensitivity of FMOC-Gly, as described below. The reaction temperature and time were also important for derivatization (Exam No. 2-4 and 2-5 in Fig. 1). Previous studies applied various conditions for the temperature and time depending on the matrix type, e.g., from room temperature to 100 °C and from 10 min to overnight, respectively.22,46,52) For the temperature condition, we selected 50 °C because the reaction efficiencies for FMOC-Gly were higher than those of 25 °C and 80 °C. In the present study, 25 °C was low for achieving a complete derivatization reaction, and there is a possibility that the target analyte decomposed thermally similar to FMOC-Glu53) for the results at 80 °C. For the reaction time, there were no significant difference in the reaction efficiencies for 20 min and 40 min. Since the short reaction time and lower temperature are considered to minimize FMOC-OH formation,50,51) 20 min was selected for the reaction time.
IDL, IQL, MDL, and MQL were determined by a similar method to that of method part 1 as described above. The obtained results are shown in Table 3. MQL by optimized conditions was comparable to or lower than that described in previous studies, as shown in Table 1.5,15,22,23,25–27) The sensitivity was improved by FMOC-Cl derivatization as indicated in previous studies.22,24)
6. Evaluation of accuracy for the overall analytical method (part 1+2)The results of evaluating the accuracy of the overall analytical method (part 1+2) at a spike level of 0.5 µg/kg and 30 µg/kg are shown in Table 4 and the obtained chromatograms of FMOC-Gly for a spike level of 0.5 µg/kg are shown in Fig. 3. The observed values were nearly 100% as the mean value for FMOC-Gly, as shown in Table 4, and the repeatability of the analysis, represented as SD, was satisfactory. Thus, these results indicate that our established method was sufficiently optimized for the analysis of Gly (FMOC-Gly). In addition, our method has superior operability because the 1.5 mL microcentrifuge tube used in method part 1 could continue to be used in method part 2 (additional apparatus is rarely required). The adsorption of Gly on glass can be reduced, which is advantageous for an easy and accurate analytical process.
| Compound | Spike level: 0.5 µg/kg | Spike level: 30 µg/kg | ||
|---|---|---|---|---|
| MM (%) | not MM (%) | MM (%) | not MM (%) | |
| Gly (FMOC-Gly) | 101.6±1.6 | 108.1±1.7 | 99.2±2.9 | 105.5±3.1 |
The values represent the mean±standard deviation; the values are described as percentages by the quantification results of IDMS relative to the spiked amount of pesticides; MM: matrix-matching calibration was used; not MM: matrix-matching calibration was not used; n=3
It is suggested that the occurrence of matrix effects has a major impact on the quantitative value. Matrix effects can cause either enhancement or suppression in the observed chromatographic response for pesticides in a matrix extract compared with the same concentration in a matrix-free solution.54) As shown in Table 4, the matrix effect was relatively small in the present study (about a 6% difference for the means of both 0.5 µg/kg and 30 µg/kg). IDMS was considered to be effective in this case since the isotope-labeled internal standard method is one of the most effective approaches for correcting the matrix effects.5,55,56) However, the matrix-matching calibration, which is also effective for canceling out the matrix effects,54,57) was used to achieve more reliable quantification because the trueness using matrix-matching calibration was slightly better than that without the matrix-matching calibration, as shown in Table 4. Our results for the matrix effect could not be compared with those of previous studies because the matrix effect of the method using MonoSpin TiO and FMOC-Cl derivatization has not been evaluated (the matrix effect for MonoSpin TiO alone was evaluated by Saito et al.30) using human serum).
7. Spike test by using another sample of human urine (sample B)The results of the spike test using sample B by the optimized method (part 1+2) are shown in Table 5. The observed values were nearly 100% as the mean value as was the case with sample A, and the results for repeatability and reproducibility were comparable or superior to those of previous studies.5,22,25,27) These results indicate that our method could perform an accurate analysis for Gly in different types of human urine samples, such as samples A and B.
| Compound | Spike level (µg/kg) | Trueness (%) | RSDr (%) | RSDR (%) |
|---|---|---|---|---|
| Gly (FMOC-Gly) | 0.5 | 104.9 | 6.3 | 6.1 |
| 30 | 101.0 | 2.5 | 2.9 |
RSDr: intra-day repeatability (n=3); RSDR: inter-day reproducibility (n=9); matrix-matching calibration was used.
The results of the spike test obtained by method part 1 and 1+2 were as follows: 98.8±4.8% for method part 1 and 103.3% ±3.5% for method part 1+2 (the values represent the mean±SD and are described as percentages of the quantification results of IDMS relative to the spiked amount of pesticides; n=3). The results for method part 1 and part 1+2 for a spike level of 1 µg/kg were in good agreement. The established method (part 1+2) was successfully validated, and the results also indicate that our method is accurate. As a side note, a matrix-matching calibration was also used for method part 1 to match the analytical conditions with those of method part 1+2 although the matrix effect was small for method part 1 (about a 1% difference for 1 µg/kg), which is similar to that of a previous study30) (provided that the matrix is different, as described above).
Optimization of the method by MonoSpin TiO extraction and FMOC-Cl derivatization with IDMS was examined and its suitability for the analysis of Gly in human urine was demonstrated. To analyze lower concentrations (e.g., <1.0 µg/kg based on previous studies5),25) as described above), Gly was derivatized by FMOC-Cl. To the best of our knowledge, FMOC-Cl derivatization combined with MonoSpin TiO and IDMS has not previously been used to analyze Gly in human urine samples. The derivatization process can be tedious and time-consuming; however, the present study showed that the reaction for the derivatization can be completed within 20 min and is advantageous for improving the sensitivity. Our established method consists entirely of simple and easy processes, e.g., the operation from beginning to end (including both MonoSpin TiO extraction and FMOC-Cl derivatization) can be performed in a 1.5 mL microcentrifuge tube. Consequently, the adsorption of Gly on glass was reduced, which can contribute to an accurate analysis. Furthermore, our method achieved excellent accuracy due to the application of IDMS. Our accurate and simple method established in this study will be useful for the analysis of many samples such as in epidemiological studies.
