Enhanced Sensitivity for Assay of Sulfonamide Drugs and ......Table 3: Summary of results for the analysis of 20 sulfonamides and trimethoprim by LCMS-8060 using QuEChERS method with

No. SSI-LCMS-073

■ Summary Twenty one sulfonamides along with trimethoprim were analyzed by LC-MS/MS using a Nexera HPLC system coupled to an LCMS-8060 triple quadrupole mass spectrometer. Diverse clean up sorbents for extraction and purification were tested using a QueChERS method. ■ Background Antibiotics present in food pose a serious public health issue. Allergies to sulfonamides are very common, and the increasing popularity of antibiotics is driving microbial resistance against current antibiotics. Monitoring the Maximum Residue Levels (MRLs) has proven essential for public health, with the MRLs for some compounds set to low ppb levels. HPLC-MS/MS is very effective at analyzing trace-level antibiotics because it is selective, fast, and very sensitive for all the compounds of interest. ■ Method Stock standard solutions of each compound were prepared by dissolving weighed amounts in alkaline methanol (5mM NH4OH) then diluting to 100 µg/mL and 1 µg/mL with mobile phase A:B 50:50. Electrospray ionization was used in positive mode. MRM parameters were optimized for each compound.

A binary gradient of water with 10% methanol, 0.3% formic acid (Pump A) and methanol with 0.3% formic acid (Pump B) was used in a Nexera UHPLC system, Table 1. A Kinetex 2.6 µm PFP 100A column (100 x 2.1mm) was used at a flow rate of 0.5 mL/min. The column temperature was set at 30ºC. 5µL of standards and samples were injected. A LCMS-8060 triple quadrupole mass spectrometer equipped with Labsolutions LCMS software and electrospray ionization (ESI) in positive mode was used for MS acquisition. Spray voltage was 4.5 kV, desolvation line temperature was 250ºC, nebulization gas was 3.0 L/min, heating gas flow 10L/min, interface temperature was 300ºC, heater block was 400 ºC, and drying gas 10 L/min. Table 2 shows concentrations at each level used to build calibration curves also used to evaluate linearity, limit of detection (LOD), limit of quantification (LOQ), and area %RSD (n=3). Except for PSA/C18 each calibration level for each QuEChERS SPE sorbent was prepared using matrix extracts from honey free of SAs and TMP (matrix-matched). Calibration levels were prepared for PSA/C18 sorbent, with HPLC buffers (without matrix in Fig.4) and using matrix-matched dilutions (with matrix in Fig.4).

Liquid Chromatography Mass Spectrometry

Enhanced Sensitivity for Assay of Sulfonamide Drugs and Trimethoprim in Honey by LCMS Triple Quadrupole Mass Spectrometry with QuEChERS Extraction

No. LCMS-073

No. SSI-LCMS-073

Recovery studies were performed with 5g honey acidified with 5mL 2.0M HCl vortexed for 1 min and sonicated for 15 minutes. Standards were spiked with each analyte with quantities ranging from 10 ppb to 50 ppb. Spiked samples were extracted with QuEChERS method using extraction salts (Supelco Supel™ QuE Citrate) with diverse clean-up sorbents following manufacturer’s procedure (Supelco Supel™ QuE PSA/C18, Zsep/C18, Zsep and Zsep+) with a final 1:5 extract dilution using methanol to inject in the LCMS system. For sulfonamide and trimethoprim quantitation, MRM transitions were optimized using a 500ng/mL mixture with 1µL infusion injections (without column) at 400uL/min. Three transitions from parent ions and fragments were selected using the optimization tool software.

Time (min) %B

0.0 2

0.5 8

5.0 30

5.01 80

5.2 5

5.5 80

6.0 80

6.01 2

8.0 2

Table 1: LC gradient time program

Concentration levels, ng/mL (ppb)

1 1000 10 2.0

2 500 11 1.0

3 250 12 0.5

4 125 13 0.24

5 62.5 14 0.12

6 31.3 15 0.06

7 15.6 16 0.03

8 7.8 17 0.015

9 3.9 18 0.0075

Table 2: Concentration levels built to define calibration curves and other parameters.

Figure 1: Chromatograms of sulfonamide drugs and trimethoprim. Standard mixture at 31.2 ng/mL (ppb) for each standard.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min

0

2500000

5000000

7500000

10000000

Sulfamerazine

Succinylsulfathiazole

Sulfadimethoxine

Sulfadiazine

Sulfamethizole

Sulfabenzamide

Sulfaguanidine

Sulfamethazine

Sulfamethoxazole

SulfaquinoxalineSulfathiazole

Sulfamethoxypyridazine

Sulfaclozine

Sulfanilamide

Sulfameter

Sulfamonomethoxine

Sulfapyridine

Sulfachloropyridazine

Sulfaphenazole

Sulfacetamide

Sulfisoxazole

No. SSI-LCMS-073

0.0 25.0 50.0 75.0 100.0 Conc.0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

45000000

Area

0 50 100 150 200 Conc.0

10000000

20000000

30000000

40000000

50000000

60000000

70000000Area

0 50 100 150 200 Conc.0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

80000000Area

0.0 25.0 50.0 75.0 100.0 Conc.0

10000000

20000000

30000000

40000000

50000000

60000000

70000000Area

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2500000

5000000

7500000

10000000

12500000

15000000

17500000

20000000

22500000

25000000

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0 50 100 150 200 Conc.0

10000000

20000000

30000000

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50000000

60000000

70000000

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5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

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0 50 100 150 200 Conc.0

25000000

50000000

75000000

100000000

125000000

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0 50 100 150 200 Conc.0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

80000000

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0 100 200 300 400 Conc.0

10000000

20000000

30000000

40000000

50000000

Area

0.0 25.0 50.0 75.0 100.0 Conc.0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

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0 100 200 300 400 Conc.0

10000000

20000000

30000000

40000000

50000000

60000000

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0 250 500 750 Conc.0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

80000000

90000000

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0.0 25.0 50.0 75.0 100.0 Conc.0

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70000000Area

0 50 100 150 200 Conc.0

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80000000

Area

Succinylsulfathiazole Sulfabenzamide Sulfacetamide

Sulfaclozine Sulfadiazine Sulfadimethoxine

Sulfaguanidine Sulfamerazine Sulfachloropyridazine

Sulfameter Sulfamethazine Sulfamethizole

r2 = 0.9991 r

2 = 0.9977 r

2 = 0.9993

r2 = 0.9998 r

2 = 0.9978

r2 = 0.9990

r2 =

0.9973

r2 =

0.9955

r2 = 0.9995 r

2 = 0.9983

r2 = 0.9987

r2 = 0.9959

Sulfamethoxazole

r2 = 0.9994

Sulfamethoxypyridazine

r2 = 0.9980

r2 = 0.9987

Sulfamonomethoxine

O

HO NH

S

O

OHN

S

N

O

O

HN

S

O

O

NH 2

O

NH

S

O

O

H 2N

S

O

O

NH

N

N

Cl

H 2N

S

O

ONH

N

N

NH2

S

O

OHN

N

N

O

O

H2N

S

O

O

NH

NH

2

NH

H 2N

S

O

O

NH

N

N

H 2N

SO

O

HN

NN

Cl

NH 2

S

O

O

HN

N

N

O

NH 2

S

O

OHN

N

N

H2N

S

O

OHN

S

NN

H2N

S

O

ONH

ON

NH2

S

O

O

HN

N

N

O

NH 2

SO

O

HN

N

N

O

NH 2

Figure 2: Representative calibration curves using standard sulfonamide mixture with matrix added. High degree of linearity was observed over the concentration range with values of r2 ≥ 0.990 for all analytes. Chemical structures are represented for each sulfonamide compound and trimethoprim.

No. SSI-LCMS-073

Figure 3: Limit of quantification LOQ calculated with MRM chromatograms for each sulfonamide and trimethoprim. Black traces correspond to MRM quantitative transition while pink represent MRM qualitative transition. LOQ, S/N and area %RDS (n=3) are described for each compound.

No. SSI-LCMS-073

A

B

Figure 4: Recovery for each compound spiked with 10 and 50 ppb of standard mixture using QuEChERS with diverse sorbents. PSA/C18 sorbent presented better performance with similar results using matrix matched and without matrix added. Only sulfonamide reported a poor recovery of 10% but showed an acceptable recovery of 58% when spiked with 50 ppb.

No. SSI-LCMS-073

Linearity

range ppb

LOQ

ppb

Linearity

range ppbR-squared

LOQ

ppb

LOQ

%RSD,

n=3

LOD

sensitivity

reduction

by matrix

effect ɫ

10ppb

Recover

y%

50ppb

Recovery

%

Succinylsulfathiazole 0.12-500 0.1 0.25-1000 0.9993 0.25 7.4 0.06 2.5 94.7 106.3

Sulfabenzamide 0.03-125 0.03 0.03-500 0.9991 0.03 10.7 <0.015 1.0 99.9 110.0

Sulfacetamide 0.03-250 0.03 0.12-500 0.9977 0.12 4.4 0.03 4.0 85.1 99.6

Sulfaclozine 0.06-500 0.06 0.25-500 0.9987 0.25 2.0 0.03 4.2 122.0 112.5

Sulfadiazine 0.015-125 0.015 0.06-250 0.9983 0.06 10.4 0.015 4.0 97.4 110.3

Sulfadimethoxine 0.03-125 0.03 0.06-250 0.9995 0.06 9.3 0.015 2.0 108.0 117.8

Sulfaguanidine 0.03-62.5 0.03 0.03-250 0.9955 0.03 5.2 <0.015 1.0 84.9 114.8

Sulfamerazine 0.03-125 0.03 0.03-250 0.9973 0.03 7.4 0.015 1.0 108.5 119.4

Sulfachloropyridazine 0.03-250 0.03 0.06-125 0.9990 0.06 4.6 0.015 2.0 99.9 108.9

Sulfameter 0.06-250 0.06 0.12-125 0.9959 0.12 8.2 0.03 2.0 107.5 114.9

Sulfamethazine 0.03-62.5 0.03 0.06-125 0.9978 0.06 2.0 0.015 2.0 110.5 122.0

Sulfamethizole 0.03-250 0.03 0.03-250 0.9998 0.03 13.4 0.015 1.0 98.6 105.7

Sulfamethoxazole 0.25-250 0.12 0.12-250 0.9994 0.12 3.1 0.06 1.0 105.0 115.2

Sulfamethoxypyridazine 0.03-125 0.03 0.06-250 0.9980 0.06 12.3 0.03 2.0 102.9 115.3

Sulfamonomethoxine 0.03-250 0.03 0.12-250 0.9987 0.12 7.5 0.03 4.0 106.0 113.0

Sulfanilamide 0.25-500 0.25 3.9-1000 0.9992 3.9 2.2 0.49 15.6 10.2 58.4

Sulfaphenazole 0.03-250 0.03 0.06-500 0.9991 0.06 2.4 0.03 2.0 110.0 117.2

Sulfapyridine 0.03-125 0.03 0.12-125 0.9980 0.12 5.6 0.03 4.0 100.9 111.3

Sulfaquinoxaline 0.015-250 0.015 0.06-125 0.9968 0.06 8.0 <0.015 4.0 99.9 108.5

Sulfathiazole 0.015-125 0.015 0.12-250 0.9967 0.12 1.4 0.03 8.0 98.5 106.4

Sulfisoxazole 0.03-500 0.03 0.12-250 0.9992 0.12 7.2 0.06 4.0 101.0 111.2

Trimethoprim 0.015-31.2 0.015 0.03-62.5 0.9974 0.03 12.5 0.03 2.0 93.0 105.4

Compound

LCMS-8060 QuEChERS SPE cleanup sorbent PSA/C18No matrix Matrix matched

ɫ Increase of LOQ values from calibration curve prepared without matrix to calibration curve prepared with matrix matched. Enhancement of LOQ for sulfanilamide with matrix matched may be related with compound instability.

Table 3: Summary of results for the analysis of 20 sulfonamides and trimethoprim by LCMS-8060 using QuEChERS method with SPE cleanup sorbent PSA/C18. ■ Results and Discussion The 8 minute HPLC gradient provided efficient separation of the sulfonamide compounds while maintaining excellent peak shape. Representative chromatograms for the sulfonamides mixture are shown in Figure 1. Authentic SAs standards were fully characterized by HPLC and MS/MS with a MRM optimized assay. Calibration curves consisting of over 17 levels, ranging from 15 ppt to 1000 ppb, are shown in Table 2. Matrix-matched calibration curves were linear with r2 > 0.990 in all QuEChERS SPE sorbents tested. Figure 3 demonstrates linearity when SPE sorbent PSA/C18 was used with diverse ranges for the antibiotics studied as seen in Table 3. Typical matrix-matched calibration curves are shown in Figure 2. Figure 3 and Table 3 demonstrate the method exhibited high sensitivity with LOQs values as low as 30 ppt detected for some SAs and TMP when matrix-matched calibration levels were prepared. When calibration curves were built without matrix interferences the LOQs dropped to as low as 15 ppt, as shown in Table 3. Criteria for LOQ selection was determined with S/N >10 calculated with rsm method; area calibration point RSD% < 15; reference ions detected with 30% allowance and area calculated accuracy >90%.

Figure 4 shows recovery of SA compounds and trimethoprim spiked in duplicate on honey with better performance for QuEChERS extractions using SPE sorbent PSA/C18, reporting a range of 85 to 122% for 10µg/kg of spiked antibiotics and a range of 99 to 122% when spiked with 50µg/kg of same compound mixture. Sulfanilamide presented a low recovery with 10% when 10µg/kg was spiked but an acceptable recovery of 58% with 50 µg/Kg, suggesting a matrix effect for lower level should be considered additional to losses involving interaction with the primary secondary amine PSA sorbent with the sulfanilamide double amino group. ■ Conclusion The combination of Nexera HPLC with LCMS-8060 triple quadrupole MS and an appropriate selection of QuEChERS SPE sorbent as extraction method provide an ideal platform for rapid, high sensitive, and selective measurement of sulfonamides and trimethoprim in honey with also an excellent recovery, improved when matrix matched calibration is used.

© Shimadzu Scientific Instruments, 2016

First Edition: May, 2016

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