di r ec t uplc_ms_ms anal ysis of amino ac ids.pdf

8/11/2019 Di r ec t UPLC_MS_MS Anal ysis of Amino Ac ids.pdf

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[ application note ][ application note ]

INTRODUCTION

The analysis of amino acids is important in a wide range of applica-

tion areas including foods, nutraceuticals, pharmaceuticals, and

various biological applications. The current methodology for amino

acid analysis is liquid chromatography (LC) with pre- or post-column

derivatization for the purposes of improving sensitivity and/or

increasing retention of the analytes of interest. While being accu-

rate, these methods are often time-consuming and labor intensive.

The direct analysis of underivatized amino acids is very attractive,

as the elimination of derivatization brings the advantages of sim-

plicity, flexibility, as well as the desired sensitivity and separation

speed. In addition, the removal of the derivatization step reduces

the possibility of altering the sample through contamination or

degradation.

This application note describes a new UPLC/MS/MS methodology

for direct amino acid analysis. The use of UltraPerformance LC

technology combined with MRM mass spectrometric detection allows

for direct amino acid analysis with high selectivity and sensitivity.

To achieve adequate retention for underivatized amino acids with

reverse-phase, UPLC columns were used with an ion pairing agent.

EXPERIMENTAL

UPLC conditions

LC system: WatersACQUITY UPLCsystem

Column: ACQUITY UPLC BEH C18,

2.1 x 50 mm, 1.7 m, 45 C

Flow rate: 0.8 mL/min (no split)

Mobile phase: A: 0.1% Pentadecafluorooctanoic

Acid (PDFOA),

99.5%:0.5% water/acetonitrile with

0.1% formic acid

B: 0.1% PDFOA,

10%:90% water/acetonitrile with

0.1% formic acid

Gradient: Time (min) %A %B Curve

0.0 99.9 0.1 6

0.5 98 2 6

2.0 80 20 6

4.0 60 40 6

4.5 0.1 99.9 6

8.0 99.9 0.1 1

The ACQUITY UPLC system with the Quattro Premier XE.

DIRECT UPLC/MS/MS ANALYSIS OF AMINO ACIDS

Peter Alden, Kate Yu, Rob PlumbWaters Corporation, Milford, MA, USA


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[ application note ]

MS conditions

MS system: Waters Quattro Premier XE

mass spectrometer

Ionization mode: Electrospray positive

Capillary voltage: 0.5 kV

Source temp.: 130 C

Desolvation temp.: 400 C

Desolvation gas: 1000 L/ hr

Cone gas flow: 50 L/hrInterscan delay: 5 ms

Interchannel delay: 10 ms

Acquisition mode: MRM

Dwell volume: Func1 Func2 Func3 Func4

20 ms 40 ms 50 ms 30 ms

Sample preparation

The amino acid separation was developed using the Pierce Amino

Acid Standard H Mix (protein hydrolysate) as a guide to optimize

resolution of acidic, basic, and neutral amino acids. The standardmixture was diluted from 1:100 to 1:500,000 in 0.1% formic acid

to generate the calibration curves.

MS/MS methods

The Quattro Premier XEs MRM transitions and optimization condi-

tions were obtained by infusing the individual amino acid standard

solutions. For the majority of the amino acids analyzed, the stron-

gest transition was the molecular ion [M+H]+minus 46, due to the

loss of formic acid from the parent amino acid.

RESULTS AND DISCUSSION

The MS method was comprised of four ESI+ functions covering the

full run time. This allowed for the maximum dwell time for each

analyte, thus giving maximum sensitivity for each amino acid. The

resulting chromatograms contained sufficient data points (>20)

across the peaks for accurate and reproducible quantitation. Figure

2 shows the MRMs obtained for the standard amino acids from all

four ESI+functions.

The best overall amino acid separation was obtained using PDFOA

as the ion pairing reagent. Even acidic and polar amino acids

(typically only weakly retained) exhibited sufficient retention for

separation and quantitation.

The optimized amino acid method utilized a 2.1 x 50 mm, 1.7 m

ACQUITY UPLC BEH C18column for high resolution, short run times,

and high throughput. The analysis required a 4.5-minute gradient

with an 8-minute total run time; longer gradients could have been

employed to further enhance the resolution.

Retention Time (minutes)0 4.53.82.51.0

Aspartic Acid

Serine

Glutamic Acid

Glycine

Threonine

Cysteine

Alanine

Histidine

Arginine

Lysine

Phenylalanine

Leucine

Isoleucine

Valine

Methionine

Proline

Tyrosine

Time0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.34

0.40

0.46

0.49

0.56

0.56

0.83

Time1.0 2.5

%

1.0 2.5

%

1.0 2.5

%

1.0 2.5

%

1.25

1.31

1.66

1.97

Time2.5 3.8

%

2.5 3.8

%

3.06

3.23

3.16

Time3.8 4.5

%

3.8 4.5

%

3.8 4.5

%

4.08

4.11

4.12

156.1> 109.9

175.2 > 70.0

146.9> 83.9

132.1> 85.8

117.8> 71.9

166.1> 119.9150.1> 103.9

115.8> 69.9

182.2> 136.0

90.0 > 43.9

121.8>75.8

120.0> 73.9

76.0 > 30.0

148.1> 83.8

106.0 > 59.9

134.1> 73.9

MS Func 1

Time0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0 4.53.82.51.0

Time0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.340.34

0.400.40

0.460.46

0.490.49

0.560.56

0.560.56

0.83

Time

0.83

Time1.0 2.5

%

1.0 2.5

%

1.0 2.5

%

1.0 2.5

%

1.0 2.5

%

1.0 2.5

%

1.0 2.5

%

1.0 2.5

%

1.251.25

1.311.31

1.661.66

1.97

Time

1.97

Time2.5 3.8

%

2.5 3.8

%

2.5 3.8

%

2.5 3.8

%

3.06

3.23

3.06

3.23

3.16

Time3.8 4.5

%

3.16

Time3.8 4.5

%

3.8 4.5

%

3.8 4.5

%

3.8 4.5

%

3.8 4.5

%

4.084.08

4.114.11

4.12

156.1> 109.9

175.2 > 70.0

146.9> 83.9

132.1> 85.8

117.8> 71.9

166.1> 119.9150.1> 103.9

115.8> 69.9

182.2> 136.0

90.0 > 43.9

121.8>75.8

120.0> 73.9

76.0 > 30.0

148.1> 83.8

106.0 > 59.9

134.1> 73.9

4.12

156.1> 109.9

175.2 > 70.0

146.9> 83.9

132.1> 85.8

117.8> 71.9

166.1> 119.9150.1> 103.9

115.8> 69.9

182.2> 136.0

90.0 > 43.9

121.8>75.8

120.0> 73.9

76.0 > 30.0

148.1> 83.8

106.0 > 59.9

134.1> 73.9

Time0.0 1.0

%

Time0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

0.0 1.0

%

MS Func 2 MS Func 3 MS Func 4

Figure 2. MRMs for all of the amino acids in the standard

mixture. Optimum sensitivity was obtained through the use

of several timed functions to maximize dwell time.


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Quantification

Figure 3 highlights four typical calibration curves (data points

in triplicate) for amino acids obtained with this method. The

responses show good linearity with detection limits ranging from

approximately 3 to 80 pg on-column. The quantification results for

the protein hydrolysate amino acid standards are summarized in

Table 1. Sensitive and linear results were obtained for nearly all

of the amino acids evaluated. Although all amino acids may be

detected using these conditions, the method is most applicable toacidic, uncharged polar and non-polar amino acids.

Figure 3. Four example calibration curves for amino acids

demonstrate the linearity and wide linear range of the

UPLC/MS/MS method.

Example calibration curves

1/500K to 1/100 dilution, 3 injections per conc.

pmol/mL

0 5000 10000 15000 20000 25000

Response

25000

50000

75000

100000

125000

150000

175000

200000

0 5000 10000 15000 20000 250000

25000

50000

75000

100000

125000

150000

175000

200000

01/20,000 to 1/100

Alanine

pmol/mL

0 5000 10000 15000 20000 25000

Response

0

200000

400000

600000

800000

1000000

1200000

0 5000 10000 15000 20000 250000

0

200000

400000

600000

800000

1000000

1200000

0

1/50,000 to 1/100

Phenylalanine

pmol/mL0 5 00 1 00 0 1 5 0 0 2 0 00 2 50 0 3 0 0 0 3 5 00 4 00 0 4 5 0 0 5 0 00

Response

0

10000

20000

30000

40000

50000

60000

70000

80000

0 5 00 1 00 0 1 5 0 0 2 0 00 2 50 0 3 0 0 0 3 5 00 4 00 0 4 5 0 0 5 0 000

0

10000

20000

30000

40000

50000

60000

70000

80000

0

1/100,000 to 1/500

Tyrosine

pmol/mL0 5 00 1 00 0 1 5 0 0 2 0 00 2 50 0 3 0 00 3 50 0 4 0 0 0 4 5 00 5 00 0

Response

0

100000

200000

300000

400000

500000

600000

0 5 00 1 00 0 1 5 0 0 2 0 00 2 50 0 3 0 00 3 50 0 4 0 0 0 4 5 00 5 00 0

0

100000

200000

300000

400000

500000

600000

1/500,000 to 1/500

Proline

Table 1. The quantification

results for the 17 amino acids

contained in the Pierce StandardH mix: LODs of 3 to 80 pg of

amino acids on-column were

obtained.

QuantificationResults

Standard Mol. Wt. T R(Min) LOD ng/mL LOD (pg) R2

Alanine 89.09 0.83 11.3 56.5 0.990

Arginine 174.2 4.12 0.871 4.35 0.887

Aspartic Acid 133.1 0.33 0.665 3.32 0.979

Cysteine 121.16 0.57 15.1 75.5 0.937

Glutamic Acid 147.13 0.45 0.735 3.67 0.947

Glycine 75.07 0.49 1.48 7.40 0.986

Histidine 155.16 4.08 0.775 3.87 0.905

Isoleucine 131.17 3.06 16.4 82 0.984

Leucine 131.17 3.23 16.4 82 0.991

Lysine 146.19 4.11 0.73 3.65 0.837

Methionine 149.21 1.66 0.746 3.73 0.992Phenylalanine 165.19 3.16 8.26 41.3 0.990

Proline 115.13 1.31 0.575 2.87 0.995

Serine 105.09 0.40 0.525 2.62 0.971

Threonine 119.12 0.56 0.595 2.97 0.980

Tyrosine 181.19 1.25 4.53 22.6 0.993

Valine 117.15 1.97 2.92 14.6 0.995


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Waters Corporation

34 Maple Street

Milford, MA 01757 U.S.A.

T: 1 508 478 2000

F: 1 508 872 1990www.waters.com

Waters Corporation

34 Maple Street

Milford, MA 01757 U.S.A.

T: 1 508 478 2000

F: 1 508 872 1990www.waters.com

CONCLUSION

A simple and rapid direct amino acid analysis method was devel-

oped that is applicable to acidic, uncharged polar, and non-polar

amino acids. High sensitivity and selectivity was achieved without

the need for pre-/post-column derivatization or split flow prior to

the MS. Typical limits of detection observed ranged from 3 to 80 pg

on-column for amino acids that work well with this method. Non-

polar amino acids tended to exhibit slightly higher LODs than other

amino acids analyzed.

Additionally, excellent linearity over a wide concentration range

was demonstrated for most of the amino acids evaluated. A sepa-

rate method1was developed to obtain optimum results with basic

amino acids.

References

1. Alden P, Yu K, Plumb R. Waters Application Note 720002003EN, Mar. 2007.

Waters, UPLC, UltraPerformance LC and ACQUITY UP LC

are registered trademarks of Waters Corporation. Quattro

Premier and The Science of Whats Possible are trademarks of

Waters Corporation. All other trademarks are the property of

their respective owners.

2007 Waters Corporation. Produced in the U.S.A.March 2007. 720002002EN. LB-PDF

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