高效液相色谱法检测培养基及微藻细胞中草甘膦、氨基甲基膦酸和抗坏血酸的含量

Juan Manuel Ostera; Valentina Olmos; Susana Puntarulo; Julián G. Bonetto; Gabriela Malanga

doi:10.21769/BioProtoc.5263

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High-Performance Liquid Chromatography Quantification of Glyphosate, Aminomethylphosphonic Acid, and Ascorbate in Culture Medium and Microalgal Cells

高效液相色谱法检测培养基及微藻细胞中草甘膦、氨基甲基膦酸和抗坏血酸的含量

Juan Manuel Ostera email

发布: 2025年04月05日第15卷第7期 DOI: 10.21769/BioProtoc.5263 浏览次数: 489

评审: Noelia ForesiPhilipp A.M. SchmidpeterWalmik Karbhari Gaikwad

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Cover of Chemosphere, featuring study using the protocol.

Jun 2020

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Abstract

Glyphosate (GLY) is a widely used herbicide that can induce oxidative stress in microalgae and other non-target organisms. The quantification of GLY in surface water is a difficult task, especially in trace-level concentrations, due to its high polarity and susceptibility to biotic and abiotic degradation. Several analytical methods have been developed for GLY quantification. Most of them use high-performance liquid chromatography (HPLC) coupled with detection by mass spectrometry (MS) and include a derivatization step to decrease the polarity of the herbicide to improve detection. This protocol describes an adaptation of an existing protocol for the quantification of GLY and its metabolite aminomethylphosphonic acid (AMPA) in a water-based microalgae culture medium using ultra-high-pressure liquid chromatography (UHPLC) coupled with fluorescence detection (FLR). The principal advantage of this protocol compared with other analytical methods that employ HPLC–MS is its low cost and accessibility since it does not require an MS detector nor radioactively labeled analytical standards. Ascorbic acid (AH^-) is one of the most important hydrosoluble non-enzymatic antioxidants in eukaryotic cells and plays a key role in many metabolic pathways of critical importance in plants and algae. In this protocol, we also describe an adaptation of a previously published protocol to quantify AH^- in blood samples to be used in microalgal cells exposed to GLY and GLY-based herbicides. The sample preparation procedure for this last protocol is fast, easy, and does not require expensive equipment. It uses an HPLC system coupled with an electrochemical detector (EC) for AH^- quantification but may be adapted to be used with a UV-Vis detector.

Key features

• These protocols employ HPLC or UHPLC. The end user should be proficient with this technique and be able to optimize chromatographic parameters as necessary.

• The GLY and AMPA quantification protocol, designed upon the method developed by Nedelkoska [1], expands its use for water-based cell culture media.

• The protocol used for AH^-, designed upon the method developed by Kutnink [2], has been modified to be used in microalgae cells.

• The quantification of AH^- employs HPLC coupled with an electrochemical detector but could be modified for its use with a UV-VIS detector.

Keywords: Glyphosate (草甘膦)

Aminomethylphosphonic acid (氨基甲基膦酸)

HPLC (HPLC)

Microalgae (微藻)

Derivatization (衍生化)

Electrochemical detector (电化学检测器)

Graphical overview

Glyphosate (GLY), aminomethylphosphonic acid (AMPA), and L-ascorbic acid (AH^-) extraction and/or derivatization. (A) Cell harvest procedure. (B) Extraction and derivatization of GLY and AMPA in cell culture medium. (C) Extraction of AH^- from microalgal cells. BBM: Bold’s basal medium. HPLC: High-performance liquid chromatography. FLR: Fluorescent detector. EC: Electrochemical detector. FMOC-Cl: 9-fluorenilmethyl chloroformate. MPA: Metaphosphoric acid.

Background

In this article, we describe two analytical protocols for the quantification of glyphosate (GLY) and ascorbic acid (AH^-) in microalgal cultures exposed to an analytical standard or a GLY-based herbicide. The first protocol described here is based on a method originally published in 2004 [1] that has been adapted to quantify GLY and its metabolite aminomethylphosphonic acid (AMPA) in a water-based culture medium. Several modifications have been made to the original method to adapt it to the equipment available in our laboratory. The main differences are related to the type of chromatographic column and mobile phase used and the derivatization step. In our protocol, several key steps of the derivatization process were identified and optimized to improve efficiency, including carefully controlling the pH of the derivatization reaction and the order in which the reagents are added. The total run time of the analysis was also improved as compared to the original method using a UHPLC system. The use of a fluorescence (FLR) detector allows this protocol to have reduced costs as it is a relatively common detector found in most standard HPLC systems. This is also the key disadvantage of our protocol because FLR detection requires a clean sample to successfully quantify the derivatized analytes. However, this method is suitable for the analysis of both GLY and AMPA in drinking and superficial water samples and has been successfully implemented for the quantification of these analytes in water-based culture media.

The second protocol described here is used to quantify ascorbate ion (AH^-) in microalgae cells. Different spectrophotometric, fluorescent, and chromatographic methods exist, which allow the determination of appreciable amounts of this antioxidant [3]. However, few methods allow direct and simultaneous detection of several chemical species. HPLC detection using different types of detection (electrochemical, fluorescent, etc.) establishes a rapid, sensitive, selective, and reproducible method for the detection of AH^- [4]. Electrochemical detection (EC) is an attractive alternative method for the detection of electroactive species because of its inherent advantages of simplicity, ease of access to miniaturization, high sensitivity, and relatively low cost. EC typically works in amperometric or coulometric mode and can be coupled with HPLC to provide high sensitivity to electroactive species [5]. The protocol described here adapts one originally published in 1987 [2] to be used with microalgal cells. It is a very simple and fast method that does not require complex processing of samples. Its main advantage (and disadvantage) is the requirement of an electrochemical detector; however, it may be replaced by a UV-VIS detector, provided that samples are thoroughly cleaned up before injection in the chromatographic system, because the pigments in microalgal and plant cells interfere with UV-VIS detectors, affecting the detection and quantification levels of this method.

Materials and reagents

Biological materials

1. Chlorella vulgaris CPCC90 (UTCC90) (Canadian Phycological Culture Centre, catalog number: CPCC90)

Reagents

GLY and AMPA analysis

1. Glyphosate analytical standard (AccuStandard, catalog number: P-015NB, CAS: 1071-83-06)

2. Aminomethyl phosphonic acid (AMPA) analytical standard (Sigma-Aldrich, catalog number: 324817, CAS: 1066-51-9)

3. Potassium dihydrogen phosphate (KH₂PO₄) for analysis EMSURE^® ISO (Supelco, catalog number: 1.04873, CAS: 7778-77-0)

4. Sodium tetraborate decahydrate (Na₂B₄O₇·10H₂O) for analysis ACS (Supelco, catalog number: 106308, CAS: 1303-96-4)

5. 9-Fluorenilmethyl chloroformate (FMOC-Cl), 99% (Sigma-Aldrich, catalog number: 23184, CAS: 28920-43-6)

6. Sodium hydroxide (NaOH), ACS reagent 97%, pellets (Sigma-Aldrich, catalog number: 221465, CAS: 1310-73-2)

7. Acetonitrile isocratic grade for liquid chromatography LiChrosolv^® (Supelco, catalog number: 100029, CAS: 75-05-8)

8. Methanol isocratic grade for liquid chromatography LiChrosolv^® (Supelco, catalog number: 106018, CAS: 67-56-1)

9. Ethyl acetate ACS grade (Merck, catalog number: 109623, CAS: 141-78-6)

10. Hydrochloric acid fuming (HCl), 37% for analysis ACS (Supelco, catalog number: 100317, CAS: 7647-01-0)

11. Orthophosphoric acid (H₃PO₄), 85% for analysis ACS (Supelco, catalog number: 100573, CAS: 7664-38-2)

12. Type 1 ultrapure water [6], resistivity 18 MΩ/cm at 25 °C)

AH^- analysis

1. Metaphosphoric acid (MPA) ACS reagent, chips, 33.5%–36.5% (Sigma-Aldrich, catalog number: 239275, CAS: 37267-86-0)

2. Type 1 ultrapure water [6], resistivity 18 MΩ/cm at 25 °C

3. L-ascorbic acid, 99% (Sigma-Aldrich, catalog number: A92902, CAS: 50-81-7)

Solutions

For GLY and AMPA quantification (see Recipes)

1. GLY stock solution

2. GLY intermediate solution 1 (GLY_I1)

3. GLY intermediate solution 2 (GLY_I2)

4. GLY intermediate solution 3 (GLY_I3)

5. AMPA stock solution

6. AMPA intermediate solution 1 (AMPA_I1)

7. AMPA intermediate solution 2 (AMPA_I2)

8. AMPA intermediate solution 3 (AMPA_I3)

9. GLY calibration curve (GLY 1; GLY 2; GLY 3)

10. AMPA calibration curve (AMPA 1; AMPA 2; AMPA 3)

11. FMOC-Cl stock solution

12. FMOC-Cl working solution

13. Sodium tetraborate decahydrate (Na₂B₄O₇·10H₂O) buffer 0.125 M pH 9

14. 3 M HCl

15. KH₂PO₄ buffer solution 50 mM pH 5.5

16. NaOH 10% w/v

17. H₃PO₄ 4% v/v

For AH^- quantification (see Recipes)

1. MPA 10% w/v

2. MPA 0.8% w/v

3. L-ascorbic acid stock solution (1 mg/mL or 1 mM in MPA 10%)

4. L-ascorbic acid working solution (1 μg/mL or 10 μM in MPA 10%)

Recipes

For GLY and AMPA quantification

1. GLY stock solution (1 mg/mL) (10 mL)

Note: Stock solutions must be stored in plastic tubes, covered with aluminum foil.

Reagent	Quantity	Final concentration	Shelf life/storage conditions
GLY analytical standard	10 mg	1 mg/mL	6 months/4 °C
Ultrapure water	10 mL
Total	10 mL

2. GLY_I1 (1 mL)

Note: The intermediate solutions must be stored in plastic tubes, covered with aluminum foil.

Reagent	Quantity	Final concentration	Shelf life/storage conditions
GLY stock solution	10 μL	10 μg/mL	48 h/4 °C
Ultrapure water	990 μL
Total	1,000 μL

3. GLY_I2(1 mL)

Note: The intermediate solutions must be stored in plastic tubes, covered with aluminum foil.

Reagent	Quantity	Final concentration	Shelf life/storage conditions
GLY_I1	10 μL	100 ng/mL	48 h/4 °C
Ultrapure water	990 μL
Total	1,000 μL

4. GLY_I3 (2.5 mL)

Note: The stock solutions must be stored in plastic tubes, covered with aluminum foil.

Reagent	Quantity	Final concentration	Shelf life/storage conditions
GLY_I2	500 μL	20 ng/mL	48 h/4 °C
Ultrapure water	2,000 μL
Total	2,500 μL

5. AMPA stock solution (1 mg/mL) (10 mL)

Note: The stock solutions must be stored in plastic tubes, covered with aluminum foil.

Reagent	Quantity	Final concentration	Shelf life/storage conditions
AMPA analytical standard	10 mg	1 mg/mL	6 months/4 °C
Ultrapure water	10 mL
Total	10 mL

6. AMPA_I1(1 mL)

Note: The intermediate solutions must be stored in plastic tubes, covered with aluminum foil.

Reagent	Quantity	Final concentration	Shelf life/storage conditions
AMPA stock solution	10 μL	10 μg/mL	48 h/4 °C
Ultrapure water	990 μL
Total	1,000 μL

7. AMPA_I2(1 mL)

Note: The intermediate solution must be stored in a plastic tube, covered with aluminum foil.

Reagent	Quantity	Final concentration	Shelf life/storage conditions
AMPA_I1	10 μL	100 ng/mL	48 h/4 °C
Ultrapure water	990 μL
Total	1,000 μL

8. AMPA_I3(2.5 mL)

Note: The intermediate solution must be stored in a plastic tube, covered with aluminum foil.

Reagent	Quantity	Final concentration	Shelf life/storage conditions
AMPA_I2	500 μL	20 ng/mL	48 h/4 °C
Ultrapure water	2,000 μL
Total	2,500 μL

9. GLY calibration curve

Note: The standard solutions for the calibration curve should be prepared in duplicate and processed on the same day as the samples. Standards of higher concentration (up to 50 ng/mL) may be prepared if necessary.

Standard solution	Reagent	Quantity	Final concentration	Shelf life/storage conditions
GLY 1	GLY_I3	50 μL	1 ng/mL	48 h/4 °C
	Ultrapure water	950 μL
	Total	1,000 μL
GLY 2	GLY_I3	250 μL	5 ng/mL	48 h/4 °C
	Ultrapure water	750 μL
	Total	1,000 μL
GLY 3	GLY_I3	500 μL	10 ng/mL	48 h/4 °C
	Ultrapure water	500 μL
	Total	1,000 μL

10. AMPA calibration curve

Standard	Reagent	Quantity	Final concentration	Shelf life/storage conditions
AMPA 1	AMPA_I3	125 μL	2.5 ng/mL	48 h/4 °C
	Ultrapure water	875 μL
	Total	1,000 μL
AMPA 2	AMPA_I3	250 μL	5 ng/mL	48 h/4 °C
	Ultrapure water	750 μL
	Total	1,000 μL
AMPA 3	AMPA_I3	500 μL	10 ng/mL	48 h/4 °C
	Ultrapure water	500 μL
	Total	1,000 μL

11. FMOC-Cl stock solution (50 mL)

Note: The solution must be stored in a plastic tube covered with aluminum foil.

Reagent	Quantity	Final concentration	Shelf life/storage conditions
FMOC-Cl 99%	0.115 g	2.3 g/mL	3 months/4 °C
Acetonitrile	50 mL
Total	50 mL	n/a

12. FMOC-Cl working solution (50 mL)

Note: The solution must be stored in a plastic tube covered with aluminum foil.

Reagent	Quantity	Final concentration	Shelf life/storage conditions
FMOC stock solution	5 mL	230 mg/L	3 months/4 °C
Acetonitrile	45 mL
Total	50 mL	n/a

13. Na₂B₄O₇·10H₂O buffer solution 0.125 M pH 9 (120 mL)

Note: The solution can be gently heated under constant stirring to dissolve the reagent completely. If precipitation is observed after some storage time, this procedure may be repeated to redissolve the precipitate. This is a very strong buffer solution; it is recommended to use 3 M HCl or another strong acid to adjust to a pH 9. The final pH of this buffer solution after preparation should be between 10 and 12.

Reagent	Quantity	Final concentration	Shelf life/storage conditions
Na₂B₄O₇·10H₂O	5.720 g	0.125 M	6 months/ambient temperature in an amber flask
Ultrapure water	Quantity sufficient
Total	120 mL	n/a

14. 3 M HCl solution (50 mL)

Reagent	Quantity	Final concentration	Shelf life/storage conditions
HCl	12.5 mL	3 M	1 year/ambient temperature in an amber flask
Ultrapure water	37.5 mL
Total	50 mL	n/a

15. KH₂PO₄ buffer solution 50 mM pH 5.5 (250 mL)

Note: It is recommended to use NaOH 10% w/v (see Recipe 16) to take this buffer to pH 5.5. This solution should be immediately filtered using a 0.22 μm nylon plain filter disc after preparation.

Reagent	Quantity	Final concentration	Shelf life/storage conditions
KH₂PO₄	1.7011 g	50 mM	1 week/4 °C in an amber flask
Ultrapure water	250 mL
Total	250 mL	n/a

16. NaOH 10% w/v solution (100 mL)

Reagent	Quantity	Final concentration	Shelf life/storage conditions
NaOH 97%	10 g	10% w/v	1 year/ambient temperature in an amber flask
Ultrapure water	100 mL
Total	100 mL

17. H₃PO₄ 4% v/v solution (100 mL)

Reagent	Quantity	Final concentration	Shelf life/storage conditions
H₃PO₄ 85%	4 mL	4% v/v	1 year/ambient temperature in an amber flask
Ultrapure water	96 mL
Total	100 mL	n/a

For AH^- quantification

1. MPA 10% w/v (25 mL)

Reagent	Quantity	Final concentration	Shelf life/storage conditions
MPA ACS reagent 33.5%–36.5%	2.5 g	10% w/v	3 months/4 °C in an amber flask
Ultrapure water	25 mL
Total	25 mL	n/a

2. MPA 0.8% w/v (200 mL)

Note: This solution should be immediately filtered using a 0.22 μm nylon plain filter disc after preparation.

Reagent	Quantity	Final concentration	Shelf life/storage conditions
MPA 10% w/v	16 mL	0.8% w/v	1 week/4 °C in an amber flask
Ultrapure water	184 mL
Total	200 mL	n/a

3. L-ascorbic acid stock solution (1 mL)

Note: The standard stock solution should be prepared on the same day as the samples.

Reagent	Quantity	Final concentration	Shelf life/storage conditions
L-ascorbic acid 99%	1 mg	1 mg/mL	24 h/4 °C
Ultrapure water	1 mL
Total	1 mL	n/a

4. L-ascorbic acid working solution

Note: The standard working solution should be prepared on the same day as the samples.

Reagent	Quantity	Final concentration	Shelf life/storage conditions
L-ascorbic acid stock solution	0.2 mL	0.1 mg/mL	24 h/4 °C
MPA 0.8% w/v	0.8 mL
Total	1 mL	n/a

Laboratory supplies

For GLY and AMPA quantification

1. Conical polypropylene centrifuge tubes with plastic screw cap, 15 mL, 120 mm × 17 mm (Sarstedt, catalog number: 62.554.009)

2. Aluminum foil

3. Microtip 2–200 μL (ABDOS, catalog number: P10101)

4. Microtip 100–1,000 μL (ABDOS, catalog number: P10102)

5. Microtip 1–5 mL (ABDOS, catalog number: P10144)

6. 1 mL disposable syringes, sterile (Neojet, catalog number: 12122)

7. Acrodisc^® nylon syringe filters, 13 mm, 0.2 μm (Waters, catalog number: WAT200524)

8. 9 mm, Amber, screw-top vial, 12 × 32 mm with cap and pre-slit PTFE/Silicone septa (Waters, catalog number: 186000847C)

9. MAGNA^® Nylon, supported plain filter disc, 0.22 μm (Osmonics Inc., catalog number: R02SP04700)

10. Chromatographic column ZORBAX Eclipse Plus C18, rapid resolution HD, 2.1 × 50 mm (1.8 μm) (Agilent, catalog number: 959757-902)

For AH^- analysis

1. Microtip 2–200 μL (ABDOS, catalog number: P10101)

2. Microtip 100–1,000 μL (ABDOS, catalog number: P10102)

3. Microtip 1–5 mL (ABDOS, catalog number: P10144)

4. Spinwin^® polypropylene microcentrifuge tubes, 1.5 mL (Tarsons, catalog number: 500010)

5. Nanosep^® centrifugal devices 0.2 μm Bio-Inert (Pall Corporation, catalog number: ODM02C34)

6. 1 mL disposable syringes, sterile (Neojet, catalog number: 12122)

7. MAGNA^® nylon, supported plain filter disc, 0.22 μm (Osmonics Inc., catalog number: R02SP04700)

8. Chromatographic column Supelcosil LC-18 3.3 cm × 4.6 mm (3 μm) (Supelco, catalog number: 58977)

9. Silica (quartz) UV-VIS spectrophotometer cuvette, 10 mm light path (Hellma, catalog number: Z600210-1EA)

Equipment

1. Refrigerated microcentrifuge (Eppendorff, model: 5415R)

2. Vortex (Willmington NC, model: 18405)

3. Automatic pipettes (1–10 μL; 2–20 μL; 20–200 μL; 100–1,000 μL; 1–5 mL) (Gilson, model: Pipetman)

4. Barnstead E-Pure water purification system (Thermo Scientific, model: D4642-33)

5. Sonicator (Branson, model: Sonifier 450)

6. HPLC with electrochemical detector (Perkin Elmer, model: Binary LC Pump 250, integrator 1022; manual injector loop 20 μL), electrochemical detector (ESA, detector model: Coulochem II; analytical cell model: 5011; guard cell model: 5020)

7. UHPLC with fluorometric detector (Waters, model: Acquity UPLC System)

8. High-precision analytical scale (Mettler, model: H6T)

9. Spectrophotometer (Beckman-Coulter, model: DU7400)

Software and datasets

1. Empower Pro (version 2154, 2005). Proprietary software from Waters Corporation. Used for equipment control and results analysis for Waters Acquity^® UPLC. Used for peak area integration. It may be replaced by any software associated with an HPLC/UHPLC that has automatic or manual peak integration capabilities

2. Microsoft Excel 365 (version 2410, 2024). It may be replaced by LibreOffice or another free and open-source datasheet software

3. BioRender (https://www.biorender.com/). The following figures were created using BioRender: Graphical overview, Ostera, J. (2025) https://BioRender.com/k98g299

Procedure

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稿件历史记录

提交日期: Dec 17, 2024

接收日期: Feb 25, 2025

在线发布日期: Mar 20, 2025

出版日期: Apr 5, 2025

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如何引用

Ostera, J. M., Olmos, V., Puntarulo, S., Bonetto, J. G. and Malanga, G. (2025). High-Performance Liquid Chromatography Quantification of Glyphosate, Aminomethylphosphonic Acid, and Ascorbate in Culture Medium and Microalgal Cells. Bio-protocol 15(7): e5263. DOI: 10.21769/BioProtoc.5263.