Amino Acid MS

PAPER www.rsc.org/methods | Analytical Methods

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

Mar�ıa Isabel Alarc�on-Flores, Roberto Romero-Gonz�alez, Antonia Garrido Frenich,* Jos�e Luis Mart�ınez Vidaland Roc�ıo Cazorla Reyes

Received 23rd April 2010, Accepted 20th August 2010

DOI: 10.1039/c0ay00263a

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.

1. Introduction

Intensive agriculture requires the use of effective fertilizers.

Therefore, the fertilizer must be composed of macro, microele-

ments, 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 interfer-

ences, repeatability, and tedious preparation steps.14 Furthermore,

Group ‘‘Analytical Chemistry of Contaminants’’, Department of AnalyticalChemistry, Almeria University, E-04071 Almeria, Spain. E-mail: [email protected]; Fax: +34950015483; Tel: +34950015985

This journal is ª The Royal Society of Chemistry 2010

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 chroma-

tography (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 elec-

trospray tandem mass spectrometry (MS/MS) was published.14

Two MS/MS transitions were monitored, minimizing back-

ground 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 ioniza-

tion modes such as high-field asymmetric waveform ion mobility

spectrometry (FAIMS) has been used in order to reduce back-

ground chemical noise and resolve isobaric interferences.21

Aminoacids can be eluted using normal phase chromato-

graphic 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

Anal. Methods, 2010, 2, 1745–1751 | 1745

http://dx.doi.org/10.1039/c0ay00263a

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reagents can be utilized, such as heptafluorobutyric acid

(HFBA),24 nonafluoropentanoic acid (NFPA) and pentadeca-

fluorooctanoic 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 chromato-

graphic 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 mm) 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, hydroxy-

proline, 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, aspara-

gine, 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

Table 1 Retention time windows (RTWs) and MS/MS parameters for the s

Compound RTW/min Cone voltage/V Collision en

Hydroxyproline 0.30–0.35 22 13Aspartic Acid 0.65–0.73 20 12Glutamic acid 1.04–1.13 19 13Glycine 1.07–1.15 19 6Lysine 1.08–1.16 20 15Glutamine 1.10–1.16 20 10Threonine 1.25–1.32 20 10Alanine 1.55–1.77 20 8Proline 1.99–2.09 22 13Tyrosine 2.20–2.26 20 13Methionine 2.55–2.60 18 10Valine 2.70–2.75 20 8Leucine 2.89–2.96 20 10Isoleucine 2.98–3.03 21 10Tryptophan 3.10–3.30 20 10Phenylalanine 3.30–3.35 20 14Asparagine 4.06–4.15 24 10Histidine 4.13–4.17 21 10Arginine 4.23–4.27 19 16

a A second transition was not monitored for these compounds.

1746 | Anal. Methods, 2010, 2, 1745–1751

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 mm 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 (Cri-

son, 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 mL.

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

elected amino acids

ergy/eV Quantification transition Confirmation transition

132.1 > 86.3 132.1 > 68.3134.0 > 70.2 134.0 > 74.2148.0 > 84.1 148.0 > 102.275.9 > 30.2 —a

147.1 > 84.1 147.1 > 130.2147.1 > 101.1 147.1 > 103.1120.0 > 74.3 120.0 > 102.190.0 > 44.2 —116.2 > 70.2 —182.1 > 136.3 182.1 > 165.2150.1 > 104.2 150.1 > 133.2118.0 > 72.2 —132.1 > 118.1 132.1 > 85.7132.2 > 69.1 132.2 > 86.1205.2 > 118.1 205.2 > 72.2166.2 > 120.3 166.2 > 103.3133.1 > 116.1 133.1 > 87.0156.1 > 110.1 156.1 > 95.1175.2 > 70.2 175.2 > 60.2



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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 desolva-

tion 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

Fig. 1 UHPLC-MS/MS chromatogram obtai


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.

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 mL of the supernatant

ned from a fertilizer spiked at 0.5 mg L�1.

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was transferred into a vial and 900 mL of acetonitrile was added.

Finally, 5 mL were injected into the UHPLC system.

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.

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 mL of formic acid, and

injected into the ESI source in positive mode at different volt-

ages. 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 quan-

tification 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 mL 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 resolu-

tion 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).

1748 | Anal. Methods, 2010, 2, 1745–1751

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.

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 mL of the

supernatant was collected and diluted with 900 mL of acetonitrile

previous to the injection onto the UHPLC. However, when this

approach was applied, only glycine, alanine, threonine, hydroxy-

proline, 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).



Table 2 Validation parameters of the developed method

Amino acid R2

Spike level (1 g kg�1) Spike level (10 g kg�1)

LOD/mg kg�1 LOQ/mg kg�1Recovery (%)a Interday precisionb Recovery (%)a Interday precisionb

Hydroxyproline 0.999 89.4 (10.9) 15.3 85.4 (7.1) 16.6 25 70Aspartic acid 0.984 103.4 (10.8) 17.2 95.4 (5.8) 13.2 3 10Glutamic acid 0.987 82.4 (6.4) 16.0 84.1 (8.8) 14.8 20 50Glycine 0.998 80.0 (10.4) 18.5 70.2 (7.2) 15.5 6 20Lysine 0.985 90.8 (7.5) 15.1 97.2 (8.9) 17.6 10 40Glutamine 0.990 94.4 (7.9) 16.3 81.4 (5.4) 16.1 50 100Threonine 0.997 92.3 (6.0) 14.1 85.0 (5.4) 12.3 20 50Alanine 0.994 71.4 (6.9) 12.3 108.6 (5.7) 16.8 50 100Proline 0.986 79.4 (5.9) 15.1 94.2 (7.7) 11.2 25 70Tyrosine 0.992 89.9 (7.4) 17.7 74.6 (10.2) 22.5 3 10Methionine 0.997 108.0 (10.8) 20.2 94.8 (6.9) 18.6 25 50Valine 0.994 86.9 (9.4) 17.2 104.9 (5.4) 16.4 20 50Leucine 0.987 90.8 (8.7) 15.7 107.5 (7.9) 13.2 20 50Isoleucine 0.996 82.4 (7.3) 12.3 108.8 (7.5) 18.3 15 50Tryptophan 0.995 78.3 (9.4) 21.3 92.3 (9.3) 13.2 10 30Phenylanine 0.999 82.6 (5.2) 13.8 98.7 (7.7) 19.2 3 10Asparagine 0.980 89.8 (7.8) 16.1 94.5 (5.8) 10.6 3 10Histidine 0.998 93.1 (7.7) 18.4 72.3 (5.3) 15.8 3 10Arginine 0.998 78.9 (6.9) 13.9 77.1 (6.0) 17.4 1 5

a Repeatability values, expressed as RSD are given in brackets (n ¼ 5). b Number of replicates: 4.

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First, matrix effects were studied to ensure bias-free analytical

results. Because the samples were not standard reference mate-

rials 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

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

Amino acid Sample A Sample B Sample C Sample D Sample

Hydroxyproline 4.0 NDa ND ND NDAspartic Acid ND 16.9 13.2 22.0 NDGlutamic acid ND 16.0 0.2 10.0 NDGlycine ND 15.2 0.4 5.8 32.0Lysine ND 0.7 11.3 7.2 24.0Glutamine ND 1.1 ND ND NDThreonine ND 6.7 1.0 ND NDAlanine 8.0 3.0 8.3 ND NDProline 5.6 21.4 5.1 4.9 NDTyrosine 0.6 2.3 0.1 ND NDMethionine 44.5 1.1 13.5 ND NDValine ND 2.9 9.1 ND NDLeucine 0.5 2.9 0.4 ND NDIsoleucine 0.8 1.2 0.3 ND NDTryptophan ND ND ND ND NDPhenylanine 2.0 3.9 0.2 ND NDAsparagine ND ND ND ND NDHistidine ND 0.5 0.3 ND NDArginine ND 3.9 0.5 4.3 NDTotal amino acids 66.0 99.7 63.9 54.2 56.0

a ND: Not detected.


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 ¼ 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 ¼ 5) were extracted

without spiking (S0) and recovery was calculated as follows: R ¼100� (S1� S0)/Cspiked. Table 2 shows the obtained results, and it

E Sample F Sample G Sample H Sample I Sample J Sample K

ND ND ND ND ND NDND ND ND ND ND ND17.7 ND ND 7.7 ND NDND ND ND ND 11.5 ND8.5 56.9 62.3 5.5 50.5 NDND ND ND 1.9 ND ND16.2 ND ND 23.5 ND NDND ND ND ND ND NDND 8.4 4.3 9.1 ND 59.0ND ND ND ND ND NDND ND ND ND ND NDND ND ND ND ND NDND ND ND ND ND NDND ND ND ND ND NDND ND ND ND ND NDND ND ND ND ND NDND ND ND ND ND NDND ND ND ND ND NDND ND ND 1.6 ND ND42.4 65.3 66.6 49.3 62.0 59.0

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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.

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.

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.

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 concentra-

tions 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).

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 inter-

ferences were detected and clean chromatograms were obtained.

1750 | Anal. Methods, 2010, 2, 1745–1751

4. Conclusions

This work presents a suitable method for the extraction, detec-

tion 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



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the proposed method convenient for the determination of the

selected amino acids in routine analysis.

Acknowledgements

RRG is grateful for personal funding through Ram�on y Cajal

Program (Spanish Ministry of Science and Innovation-European

Social Fund).

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Amino Acid MS