Rapid determination of underivatized amino acids in fertilizers by ultra high performance liquid chromatography coupled to tandem mass spectrometry

Abstract

The analysis of 19 underivatized protein amino acids by ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) is studied. Amino acids were separated by reversed phase, adding to the mobile phase pentadecafluorooctanoic acid as ion pairing reagent. The selected amino acids were eluted in less than 8 min. MS/MS parameters were optimized, using electrospray ionization (ESI) in positive mode for the detection of the amino acids. A simple solid-liquid extraction with water and heptafluorobutyric acid was used for the extraction of the compounds from the fertilizer. All amino acids were extracted with recoveries higher than 70% and relative standard deviation (RSD) lower than 22.5% (inter-day precision). Limits of quantification were always lower than 100 mg kg⁻¹ of sample, for all the compounds. The validated method is simple, fast and sensitive and it has been applied for the determination of amino acids in fertilizers.

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1. Introduction

Intensive agriculture requires the use of effective fertilizers. Therefore, the fertilizer must be composed of macro, microelements, physiologically active substances, growth stimulants and organic molecules such as amino acids.¹ The use of amino acids is often recommended for critical conditions during plant growth (i.e., after transplantation, in the flowering period), and also at climatic stresses (night frosts, drought) or plant diseases. These compounds are particularly effective when they are used in combination with other microelements in fertilizers.^2,3

The determination of amino acids is very important in food, biological fluids, fermentation products and fertilizers, because these molecules play an important function in nutritional quality of food and beverages and in the control of samples fortified with proteins.^4,5

Up to now, there are different methods to separate and detect amino acids in fertilizers. Most of the chromatographic methods include a pre- or post-column derivatization,⁶ using several derivatization reagents depending on the type of detection. Thus, fluorescein isothiocyanate (FITC)⁷ and o-phthaldialdehyde/alkylthiols (OPA/R-SH),⁸ have been used when fluorescence detection is applied, whereas phenyl isothiocyanate (PITC)⁹ and dimethylaminoazobenzenesulfonyl chloride, commonly named as dabsyl chloride, (Dbs-Cl)¹⁰ are only used with UV detection. However other derivatizating reagents such as dansyl chloride (Dns-Cl),¹¹ 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)¹² and 9-fluorenylmethyl chloroformate (FMOC),¹³ can be used with either fluorescence or UV detection.

However, this derivatization process involves several problems such as instability of the derivatized compounds, reagent interferences, repeatability, and tedious preparation steps.¹⁴ Furthermore, some reagents are not able to derivatize the secondary amino acids.¹⁵ For this reason, in the last few years, derivatization process has been avoided and underivatized amino acids have been determined in order to reduce sample handling and increase sample throughput.⁶

Most of the available detectors coupled with liquid chromatography (LC) have been used for the analysis of underivatized amino acids, such as electrochemical,¹⁶ UV¹⁷ and amperometric detection.¹⁸ However, some of these methods have several drawbacks such as low sensitivity, baseline drift, incompatibility with gradient elution mode and unreliable results were obtained when complex matrices had been analyzed.

Other methods based on evaporative light scattering (ELS)¹⁹ and mass spectrometry (MS)²⁰ detection are also used. MS is usually selected because it also provides structural information. Moreover, when MS is used as the detection method, it can provide unambiguous evidence of amino acid identification and therefore complete resolution of the selected compounds is not highly demanding, simplifying the chromatographic step.

Despite of the advantages of MS, little work has been published using this type of detection.^6,14,21,22 For instance, the analysis of underivatized protein amino acids by LC and electrospray tandem mass spectrometry (MS/MS) was published.¹⁴ Two MS/MS transitions were monitored, minimizing background noise, and increasing the sensitivity in comparison with single MS mode. Although conventional electrospray ionization has been used for the analysis of 20 amino acids,⁶ other ionization modes such as high-field asymmetric waveform ion mobility spectrometry (FAIMS) has been used in order to reduce background chemical noise and resolve isobaric interferences.²¹

Aminoacids can be eluted using normal phase chromatographic columns, which allow good separation of amino acids and is compatible with MS detection.⁶ However, if reversed-phase LC is applied, it is necessary to use ion-pair agents for a good elution of underivatized amino acids due to the high polarity of these compounds.²³ Furthermore, if MS/MS is used as the detection technique volatile ion pairs can be used, and several reagents can be utilized, such as heptafluorobutyric acid (HFBA),²⁴ nonafluoropentanoic acid (NFPA) and pentadecafluorooctanoic acid (PDFOA). This provides the best overall retention characteristics for amino acids despite accumulation in the column.²²

Another important topic during the determination of amino acids is the reduction in the analysis time during the chromatographic step. For that purpose, the introduction of ultra high performance liquid chromatography (UHPLC) has decreased the analysis time by reducing the particle size of the stationary phase (<2 μm) providing significant advantages in relation to conventional LC, such as increased speed of analysis, resolution, sensitivity and peak capacity.²⁵ Despite the advantages of UHPLC, up to now it has not been tested for the analysis of amino acids.

This work proposes the use of UHPLC coupled to MS/MS for the identification and quantification of 19 underivatized essential amino acids, namely lysine, arginine, histidine, glycine, alanine, valine, isoleucine, leucine, threonine, asparagine, methionine, glutamine, phenylalanine, proline, tyrosine, tryptophan, hydroxyproline, glutamic acid and aspartic acid in fertilizers, with a run time less than 10 min in order to increase sample throughput. The proposed method is fast and it can be applied in routine analysis.

2. Experimental

2.1 Chemicals and reagents

Commercial amino acids standards (lysine, arginine, histidine, glycine, alanine, valine, isoleucine, leucine, threonine, asparagine, methionine, glutamine, phenylalanine, proline, tyrosine, tryptophan, hydroxyproline, glutamic acid and aspartic acid) were supplied by Fluka (Steinheim, Germany). Stock standard solutions of individual compounds (with concentrations between 600 and 700 mg L⁻¹) were prepared by exact weighing of the powder and dissolved in 50 mL of a solution of hydrochloric acid (HCl) 0.05 M (J.T. Baker, Deventer, Holland), which were then stored at 4 °C in brown bottles. A multicompound working standard solution at a concentration of 10 mg L⁻¹ of each compound was prepared by appropriate dilutions of the stock solutions with HCl 0.05 M and stored in screw-capped glass tubes at 4 °C. HPLC-grade acetonitrile (ACN) was supplied by J.T. Baker.

Pentadecafluorooctanoic acid (PDFOA), heptafluorobutyric acid (HFBA) and formic acid (purity >98%) were obtained from Sigma (Madrid, Spain). Ultrapure water was obtained from a Milli-Q Gradient water system (Millipore, Bedford, MA, USA).

2.2 Apparatus and software

Chromatographic analyses were performed using an Acquity UPLC system (Waters, Milford, MA, USA), and separations were achieved using an Acquity UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm particle size) from Waters. Mass spectrometry analysis was carried out using a Waters Acquity TQD tandem quadrupole mass spectrometer (Waters, Manchester, UK). The instrument was operated using electrospray ionization (ESI). Data acquisition was performed using MassLynx 4.0 software with QuanLynx program (Waters).

Centrifugations were performed in a high-volume centrifuge from Centronic (Barcelona, Spain). A pH meter, GLP 21 (Crison, Barcelona, Spain) was also used.

2.3 UHPLC-MS/MS analysis

Chromatographic analyses were carried out with a mobile phase consisting of acetonitrile (eluent A) and an aqueous solution of PDFOA (0.12% w/v) and formic acid (0.05% v/v) (eluent B) at a flow rate of 0.8 mL min⁻¹. Column temperature was kept at 45 °C and the injection volume was 5 μL.

The gradient profile was as follows: the initial mobile composition was 0% of eluent A and it was increased to 2% in 0.5 min. After that, it was increased to 20% in 1.5 min and then increased to 40% in 2 min. Finally it was increased to 100% A in 0.5 min and this composition was kept constant for 2 min, before being returned to the initial conditions in 0.5 min, keeping this composition 1 min prior the next analysis, obtaining a total run time of 8 min.

All amino acids were detected using ESI in positive mode. The capillary voltage and the extractor voltage were 3 kV and 2 V, respectively. The source temperature was 130 °C and desolvation temperature 350 °C. The cone gas (nitrogen) and desolvation gas (also nitrogen) were set at flow rates of 80 L h⁻¹ and 600 L h⁻¹ respectively, and the collision-induced dissociation was performed using argon as the collision gas at the pressure of 4 × 10⁻³ mbar in the collision cell. The specific MS/MS parameters for each amino acid are shown in Table 1.

Table 1 Retention time windows (RTWs) and MS/MS parameters for the selected amino acids

Compound	RTW/min	Cone voltage/V	Collision energy/eV	Quantification transition	Confirmation transition
a A second transition was not monitored for these compounds.
Hydroxyproline	0.30–0.35	22	13	132.1 > 86.3	132.1 > 68.3
Aspartic Acid	0.65–0.73	20	12	134.0 > 70.2	134.0 > 74.2
Glutamic acid	1.04–1.13	19	13	148.0 > 84.1	148.0 > 102.2
Glycine	1.07–1.15	19	6	75.9 > 30.2	—^a
Lysine	1.08–1.16	20	15	147.1 > 84.1	147.1 > 130.2
Glutamine	1.10–1.16	20	10	147.1 > 101.1	147.1 > 103.1
Threonine	1.25–1.32	20	10	120.0 > 74.3	120.0 > 102.1
Alanine	1.55–1.77	20	8	90.0 > 44.2	—
Proline	1.99–2.09	22	13	116.2 > 70.2	—
Tyrosine	2.20–2.26	20	13	182.1 > 136.3	182.1 > 165.2
Methionine	2.55–2.60	18	10	150.1 > 104.2	150.1 > 133.2
Valine	2.70–2.75	20	8	118.0 > 72.2	—
Leucine	2.89–2.96	20	10	132.1 > 118.1	132.1 > 85.7
Isoleucine	2.98–3.03	21	10	132.2 > 69.1	132.2 > 86.1
Tryptophan	3.10–3.30	20	10	205.2 > 118.1	205.2 > 72.2
Phenylalanine	3.30–3.35	20	14	166.2 > 120.3	166.2 > 103.3
Asparagine	4.06–4.15	24	10	133.1 > 116.1	133.1 > 87.0
Histidine	4.13–4.17	21	10	156.1 > 110.1	156.1 > 95.1
Arginine	4.23–4.27	19	16	175.2 > 70.2	175.2 > 60.2

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2.4 Extraction procedure

All fertilizer samples were processed according to the following procedure: an aliquot of fertilizer (0.5 g) was weighed and 10 mL of water solution at pH 1.5 set with HFBA was added. The mixture was vortexed (1 min) and centrifuged for 15 min at 3000 rpm (1489 g). After centrifugation, 100 μL of the supernatant was transferred into a vial and 900 μL of acetonitrile was added. Finally, 5 μL were injected into the UHPLC system.

3. Results and discussion

3.1 Optimization of the analytical method

Chromatographic and MS conditions were optimized in order to get suitable sensitivity and reduced analysis time. First, ESI-MS/MS parameters were optimized by direct infusion of a standard solution of 20 mg L⁻¹ of each amino acid at a flow rate of 0.01 mL min⁻¹. The solution was prepared in 5 mL of a mixture of methanol–water (50 : 50, v/v) and 50 μL of formic acid, and injected into the ESI source in positive mode at different voltages. Full scan spectra and the MS/MS spectra were acquired. First, the cone voltage was optimized in single MS mode in order to obtain the most abundant precursor ion, which was the protonated molecule [M–H]⁺ for all the amino acids. From the collision induced dissociation (CID) spectra, the collision energy was optimised, selecting the most sensitive transition for quantification purposes. Table 1 shows the MS/MS transitions as well as the cone voltages and collision energies optimised for each amino acid. It must be emphasized that the same collision energy was applied for the two transitions monitored for each compound. It can be observed that most amino acids show an abundant product ion at [M + H-46]⁺, which corresponds to the neutral loss of formic acid by a rearrangement,²² whereas some compounds such as asparagine, lysine, methionine, and tyrosine have a common neutral loss of m/z 17 due to the loss of NH₃.

For most of the compounds, two transitions were monitored for each amino acid, except for glycine, proline, alanine and valine. Because these compounds have low molecular weight, only one selective and sensitive transition was obtained and monitored for further experiments (see Table 1).

Then, the chromatographic conditions were studied to obtain the best peak shape and reduce analysis time. Several gradient profiles were studied, obtaining good response with the gradient described in the Experimental Section. Other parameters such as column temperature, flow rate and injection volume were studied in order to get a fast and reliable separation, obtaining the best results when 45 °C was used as column temperature, 0.8 mL min⁻¹ as flow rate and 5 μL were injected onto the chromatographic system. Bearing in mind that PDFOA can be accumulated in the column,²² modifying the retention time of the compounds, after each batch the column was flushed with 100% of acetonitrile at 0.5 mL min⁻¹ for 30 min to overcome this problem.

Fig. 1 shows a representative multiple reaction monitoring (MRM) chromatogram obtained from a standard mixture of the selected amino acids at 0.5 mg L⁻¹. As can be seen, complete resolution for all the amino acids is not achieved, but the use of MS/MS allows the analysis without chromatographic resolution between compounds. Furthermore, the application of the chromatographic technique also allows the discrimination between isobaric compounds. For instance, hydroxyproline and isoleucine have the same precursor ion and fragmentation pattern (see Table 1). However, they have different retention times and therefore they can be determined separately. On the other hand, isoleucine and leucine have the same retention time and precursor ion, as well as common product ion (m/z 86). However, for leucine an ion at m/z 118 is obtained (see Fig. 2), whereas this ion was not obtained for isoleucine, and it was used for quantification purposes, bearing in mind that isolecine can not interfere. In the case of isoleucine, an ion at m/z 69 was obtained, which was not observed for leucine, and therefore, it was used for quantification, despite the lower intensity.

Fig. 1 UHPLC-MS/MS chromatogram obtained from a fertilizer spiked at 0.5 mg L⁻¹.

Fig. 2 MS/MS spectrum obtained in ESI positive mode at collision energy of 10 eV and precursor ion of 132.2 of: (a) isoleucine and (b) leucine.

Finally, there is another pair of compounds with the same molecular (m/z 147) mass and retention time: glutamine and lysine. However, they have different product ions (see Table 1) and reliable determination of this pair of compounds can be carried out.

For the extraction of amino acids from fertilizers, a method based on the extraction of amino acids with a solution of sodium chloride was used.²⁶ Four grams of fertilizer was weighed and 25 mL of 0.5 M sodium chloride was added. Then, the mixture was centrifuged at 5000 rpm during 10 min. Then, 100 μL of the supernatant was collected and diluted with 900 μL of acetonitrile previous to the injection onto the UHPLC. However, when this approach was applied, only glycine, alanine, threonine, hydroxyproline, isoleucine, asparagine, glutamic acid, phenylalanine, arginine, tyrosine and tryptophan present recoveries higher than 70%, whereas for the rest of amino acid recoveries ranged from 30 to 60%. In order to improve the extraction, an acidic solution was prepared with HFBA, which can form ion pairs with amino acids and the extraction of these compounds can be improved. When the extraction procedure described in the Experimental Section was applied, better recovery values were obtained, and they ranged from 70 to 110% for the assayed compounds.

3.2 Validation of the proposed method

The selected analytical method was validated in terms of linearity, trueness, repeatability (intraday precision), limits of detection (LOD) and limits of quantification (LOQ).

First, matrix effects were studied to ensure bias-free analytical results. Because the samples were not standard reference materials and no blank fertilizer samples were available, fertilizer samples were spiked, before extraction, with the amino acids at different concentrations (from 0.5 to 2 g kg⁻¹), and the slopes of the calibration plots were compared with results obtained when the whole process was applied to standard solutions of the amino acids. The calibration curve obtained using spiked fertilizer was not significantly different from that obtained by use of standard solutions and external calibration was used for quantification.

Then, linearity of the response was evaluated by injecting five concentrations of the selected amino acids (from 0.1 to 2 mg L⁻¹). The calibration functions obtained by plotting the peak area versus the concentration of the compound were linear, with the determination coefficient higher than 0.98 for all compounds (see Table 2). For that purpose, and bearing in mind that some unsymmetrical peaks can be obtained due to the lower retention time of some compounds (Fig. 1), automatic quantification was revised and when it is not reliable, manual integration was carried out. Trueness was estimated through recovery studies. Before extraction, different aliquots of fertilizer (n = 5) were spiked at two levels, 1 and 10 g kg⁻¹, with the target compounds and were extracted with the developed method (S1). On the other hand, other aliquots of the same fertilizer sample (n = 5) were extracted without spiking (S0) and recovery was calculated as follows: R = 100 × (S1 − S0)/C_spiked. Table 2 shows the obtained results, and it can be observed that recoveries ranged from 72.3 (histidine) to 108.8% (isoleucine) for the selected compounds at 1 g kg⁻¹ and from 70.2 (glycine) to 108.0% (methionine) at 10 g kg⁻¹.

Table 2 Validation parameters of the developed method

Amino acid	R^2	Spike level (1 g kg^−1)		Spike level (10 g kg^−1)		LOD/mg kg^−1	LOQ/mg kg^−1
		Recovery (%)^a	Interday precision^b	Recovery (%)^a	Interday precision^b
a Repeatability values, expressed as RSD are given in brackets (n = 5). b Number of replicates: 4.
Hydroxyproline	0.999	89.4 (10.9)	15.3	85.4 (7.1)	16.6	25	70
Aspartic acid	0.984	103.4 (10.8)	17.2	95.4 (5.8)	13.2	3	10
Glutamic acid	0.987	82.4 (6.4)	16.0	84.1 (8.8)	14.8	20	50
Glycine	0.998	80.0 (10.4)	18.5	70.2 (7.2)	15.5	6	20
Lysine	0.985	90.8 (7.5)	15.1	97.2 (8.9)	17.6	10	40
Glutamine	0.990	94.4 (7.9)	16.3	81.4 (5.4)	16.1	50	100
Threonine	0.997	92.3 (6.0)	14.1	85.0 (5.4)	12.3	20	50
Alanine	0.994	71.4 (6.9)	12.3	108.6 (5.7)	16.8	50	100
Proline	0.986	79.4 (5.9)	15.1	94.2 (7.7)	11.2	25	70
Tyrosine	0.992	89.9 (7.4)	17.7	74.6 (10.2)	22.5	3	10
Methionine	0.997	108.0 (10.8)	20.2	94.8 (6.9)	18.6	25	50
Valine	0.994	86.9 (9.4)	17.2	104.9 (5.4)	16.4	20	50
Leucine	0.987	90.8 (8.7)	15.7	107.5 (7.9)	13.2	20	50
Isoleucine	0.996	82.4 (7.3)	12.3	108.8 (7.5)	18.3	15	50
Tryptophan	0.995	78.3 (9.4)	21.3	92.3 (9.3)	13.2	10	30
Phenylanine	0.999	82.6 (5.2)	13.8	98.7 (7.7)	19.2	3	10
Asparagine	0.980	89.8 (7.8)	16.1	94.5 (5.8)	10.6	3	10
Histidine	0.998	93.1 (7.7)	18.4	72.3 (5.3)	15.8	3	10
Arginine	0.998	78.9 (6.9)	13.9	77.1 (6.0)	17.4	1	5

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Precision of the overall method was estimated by performing both repeatability and reproducibility (inter-day precision). Repeatability was evaluated at the two concentration levels of the recovery studies, performing five replicates at each level (Table 2). It can be noted that repeatability values (expressed as relative standard deviation, RSD) were always lower than 11%. Inter-day precision was evaluated at the same concentration levels in four different days (see Table 2), obtaining values lower than 20% for the two levels assayed, except for tryptophan (21.3%), tyrosine (22.5%) and methionine (20.2%).

LODs and LOQs were determined as the lowest concentration level that yielded a signal-to-noise (S/N) ratio of 3 and 10 (when the quantification ion was monitored), and they are shown in Table 2. Bearing in mind the presence of matrix effect and no “blank” matrices were available, LODs and LOQs were estimated by extrapolation of the S/N of the extract with known amount of analytes and they were expressed as mg kg⁻¹ of sample (fertilizer). LODs ranged from 1 mg kg⁻¹ (arginine) to 50 mg kg⁻¹ (alanine and glutamine) whereas LOQs ranged from 5 mg kg⁻¹ (arginine) to 100 mg kg⁻¹ (alanine and glutamine). These were sufficient for quantification of the compounds in real samples.

Finally, identification of the amino acids was carried out by searching in the appropriate retention time windows (RTWs), defined as the mean retention time ± three standard deviations of the retention time of ten samples spiked at 100 mg kg⁻¹ for each compound (Table 1). For all cases, the variability on the retention time was lower than 5%. In general, confirmation was carried out by comparison of the signal intensity ratios of the two transitions (quantification and confirmation) with those obtained using fortified fertilizer samples.

3.3 Analysis of fertilizers

The validated method was applied to the determination of amino acids in 11 different commercial fertilizers. Internal quality control was applied in every batch of samples in order to check if the system is under control. This quality control was based on the evaluation of the recovery in one sample spiked at 1 g kg⁻¹, as was indicated previously, and it is also based on the evaluation of the linearity in the working concentration range.

The obtained results are shown in Table 3. It can be observed that there are differences among the individual content of each amino acid in each analyzed sample. However, lysine and proline were the most frequently detected compounds, with concentrations ranging from 0.7 to 62.3 g kg⁻¹ and 5.1 to 59.0 g kg⁻¹, respectively. On the other hand, hydroxyproline was only detected in one sample (Sample A), showing a concentration of 4.0 g kg⁻¹, whereas asparagine was not detected in any sample. In relation to the total content of amino acids, the total concentration ranged from 42 to 66 g kg⁻¹, except for sample B, which shows the higher concentration (99 g kg⁻¹).

Table 3 Amino acid concentration (g kg⁻¹) in the analyzed fertilizers

Amino acid	Sample A	Sample B	Sample C	Sample D	Sample E	Sample F	Sample G	Sample H	Sample I	Sample J	Sample K
a ND: Not detected.
Hydroxyproline	4.0	ND^a	ND	ND	ND	ND	ND	ND	ND	ND	ND
Aspartic Acid	ND	16.9	13.2	22.0	ND	ND	ND	ND	ND	ND	ND
Glutamic acid	ND	16.0	0.2	10.0	ND	17.7	ND	ND	7.7	ND	ND
Glycine	ND	15.2	0.4	5.8	32.0	ND	ND	ND	ND	11.5	ND
Lysine	ND	0.7	11.3	7.2	24.0	8.5	56.9	62.3	5.5	50.5	ND
Glutamine	ND	1.1	ND	ND	ND	ND	ND	ND	1.9	ND	ND
Threonine	ND	6.7	1.0	ND	ND	16.2	ND	ND	23.5	ND	ND
Alanine	8.0	3.0	8.3	ND	ND	ND	ND	ND	ND	ND	ND
Proline	5.6	21.4	5.1	4.9	ND	ND	8.4	4.3	9.1	ND	59.0
Tyrosine	0.6	2.3	0.1	ND	ND	ND	ND	ND	ND	ND	ND
Methionine	44.5	1.1	13.5	ND	ND	ND	ND	ND	ND	ND	ND
Valine	ND	2.9	9.1	ND	ND	ND	ND	ND	ND	ND	ND
Leucine	0.5	2.9	0.4	ND	ND	ND	ND	ND	ND	ND	ND
Isoleucine	0.8	1.2	0.3	ND	ND	ND	ND	ND	ND	ND	ND
Tryptophan	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Phenylanine	2.0	3.9	0.2	ND	ND	ND	ND	ND	ND	ND	ND
Asparagine	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Histidine	ND	0.5	0.3	ND	ND	ND	ND	ND	ND	ND	ND
Arginine	ND	3.9	0.5	4.3	ND	ND	ND	ND	1.6	ND	ND
Total amino acids	66.0	99.7	63.9	54.2	56.0	42.4	65.3	66.6	49.3	62.0	59.0

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Finally, Fig. 3 shows the obtained chromatograms of a fertilizer (sample B) containing isoleucine, glutamic acid and threonine at 1.2, 16.0 and 6.7 g kg⁻¹ respectively. It can be observed that no interferences were detected and clean chromatograms were obtained.

Fig. 3 UHPLC-MS/MS chromatogram for a fertilizer containing: (a) isoleucine at 1.2 g kg⁻¹, (b) glutamic acid at 16.0 g kg⁻¹ and (c) threonine at 6.7 g kg⁻¹.

4. Conclusions

This work presents a suitable method for the extraction, detection and quantification of 19 underivatized amino acids in fertilizers by UHPLC-MS/MS. The use of volatile ion pairing reagents and reversed phase allows a suitable separation of the compounds in a reasonable time (less than 8 min), and detection by MS/MS, avoiding the interferences of the selected analytes with each other, reducing analysis time in comparison with current analytical methods. Furthermore, the derivatization step is also avoided and the method is selective and sensitive. The developed method combines the high-resolution, capacity and fast analysis of UHPLC-MS/MS with a rapid extraction process, allowing a simple, rapid and reliable analysis of amino acids, increasing sample throughput. Validation parameters such as trueness, precision and LOQs were satisfactory and they make the proposed method convenient for the determination of the selected amino acids in routine analysis.

Acknowledgements

RRG is grateful for personal funding through Ramón y Cajal Program (Spanish Ministry of Science and Innovation-European Social Fund).

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