Analysis of sulfonamides in the low pg range by capillary LC using diode array and mass spectrometric detection

Rainer Schuster Christine Miller

Abstract Sulfonamides are a class of drugs with antibacterial properties that are widely used to treat a variety of infections. The sulfonamide drugs exhibit amphoteric properties due to the acidic N-H linkage adjacent to the sulfonyl group and to the basic character of the para-NH2 group. These drugs can be easily detected in either positive or negative ion mode using atmospheric pressure ionization (API) LC/MS. For electrospray LC/MS, the mobile phase was acidified with 0.1 % formic acid to improve positive ion formation. Studies have shown that electrospray behaves as a concentration-sensitive detector similar to UV-Vis detectors. For concentration- sensitive LC detectors, reducing column diameter will increase sensitivity. For example, changing from a 4.6-mm to a 0.5-mm column will give approximately a 100-fold increase in sensitivity with the same injection volume. Much lower injection volumes can be used to achieve the same peak height. The Agilent 1100 Series capillary LC system can accurately and reproducibly inject sample volumes of 100 nl and less with a newly developed autosampler. Using sulfonamides as typical small molecule analytes, excellent data was achieved for precision, linearity and repeatability from 100-nl injections on a 0.5-mm capillary column. With capillary LC using diode-array detection (capLC-DAD), less than 10 pg on-column was detected. By combining the capillary LC system with the high sensitivity of selected ion monitoring (SIM) on a single quadrupole mass spectrometer, less than 1 pg on-column was detected. This excellent sensitivity is a major advantage of the capillary LC concept. Conditions Experimental An Agilent 1100 Series capillary LC system and an Agilent 1100 Series Sample: sulfonamide mixture LC/MSD SL with Agilent ChemStation for instrument control, data Column: ZORBAX SB C18, 150 0.5 mm,3.5 m acquisition and analysis was used for all experiments. The LC/MSD Mobile phase: A = 0.1 % formic acid in water was used with electrospray ionization and with the capillary LC B = 0.1 % formic acid in acetonitrile nebulizer that is optimized for capillary LC flow rates. The diode-array Flow rate: 13 L/min was equipped with a 500-nl volume, 10-mm path-length flow cell. The Gradient: at 0 min 15 % B, analytical column was a Zorbax SB-C18 150 0.5 mm, 3.5 m. All at 10 min 95 % B, at 11 min 15 % B chemicals were of HPLC grade or better. The sulfa drug mixture was Injection volume: 100 nL prepared from aqueous 10 mg/l stock solutions. Column compartment temperature: 25 C Diode array detection 1 sulfanilamide 3 sulfadiazine 5 sulfamethazine 7 sufameter 278/10 nm (reference 450/80 nm)
2 sulfacetamide 4 sulfapyridine 2 1 3 100 pg 10 pg 4 6 sulfamerazine 5 6 7 Absorbance [mAU] 4 3 2 1 0 -1 0 2 4 6 8 10 [min]

Figure 1 Analysis of a mixture of 7 sulfonamides with diode array at 10 pg and 100 pg absolute with 100-nL injection volume.

MS conditions Source: ESI Ionization mode: positive Vcap: 4000 V Nebulizer: 10 psig Drying gas flow: 7 l/min Drying gas temperature: 150 C SIM ions: 0 min: 172.9 (sulfanolamide) and 215.1 (sulfaguanidine), 3.6 min: 214.9 (sulfacetamide), and 251.1 (sulfadiazine), 5.6 min: 250.1 (sulfapyridine), 279.1 (sulfamethazine) and 281.1

Results Figure 1 shows the analysis of seven sulfonamides with 100 pg and 10 pg on-column with diode-array detection. With a signal-to-noise ratio of 3, the 10 pg injection is at the detection limit. Figure 2A shows the MS signal (total ion chromatogram) for the same analysis. The injected amounts for MS are 10 pg and 1 pg (10 times less than with DAD). Several of the sulfonamides elute closely and extracting just the ion of interest allows for better integration and calibration, especially at low levels. In addition, using extracted ion chromatograms (EICs) can enhance the visibility of smaller components in a mixture. Figure 2B illustrates the use of EICs for three of the components of the sulfonamide mixture at 1 pg on-column. Relative standard deviation for the sulfonamides was < 0.1 % for retention time and < 2.8 % for area respectively, based on ten runs with 100 pg and 100 nl injection volumes (table 1). The assay was linear in a range from 1 pg/l to 1 ng/l (r2 >0.9999) for the MSD and 10 pg/l - 100 ng/l for DAD. Even when dealing with extremely small sample volumes, the capillary LC system is capable of giving highly reproducible quantitative results and which makes the system ideal in situations where sample volumes are limited and multiple analyses are necessary on a single sample. A combined capillary LC/MS system enhances the sensitivity and specificity of the analysis.
Sulfameter Run # Mean S.D RSD RetTime [min] 8.083 4.69e-3 0.058 Sulfameter Mean S.D. RSD 8.346 4.54e-3 0.054 Signal: DAD1 A, Sig=278,20 Ref=450,80 Amount [pg} 118.6 2.01 1.70 Area [mAU*s] 21.96 0.35 1.62 Height [mAU] 3.41 0.044 1.29 Width 0.099 8 e-4 0.857 Symm.[min] 0.83 0.01 1.70

Equipment
Agilent 1100 Capillary LC system consisting of Capillary pump Micro vacuum degasser Thermostatted column compartment Micro autosampler Diode array detector, 500 nL flow cell Agilent ChemStation Agilent 1100 Series LC/MSD SL Electrospray source Capillary LC nebulizer Column ZORBAX SB C-18, 150 0.5 mm, 3.5 m (Agilent p/n 5064-8262)

Signal: MSD1 TIC, MS File) 116.9 1.87 1.60 2.46e5 3943 1.60 2.47e4 358 1.44 0.1495 3.40e-3 2.27 0.81 0.01 1.46

Table 1: RSD values for DAD and MSD

B
1 2 3 4 sulfaguanidine sulfanilamide sulfacetamide sulfadiazine 5 6 7 8 sulfapyridine sulfamerazine sulfamethazine sufameter
2500 2200 2200 sulfamethazine m/z 279.1 sulfacetamide m/z 215.1

A
Abundance 5000

7 1 5 6 2 3 10 pg 0 1 pg
2

600 1600

sulfameter m/z 281.1 min

4000 3000 2000

1000

400

Rainer Schuster is an application chemist at Agilent Technologies, Waldbronn, Germany. Christine Miller is an application chemist at Agilent Technologies in Palo Alto, CA For more information on our products and services, visit our worldwide website at http://www.agilent.com/chem

10 min

Figure 2 Analysis of the standard mix with 10 pg and 1 pg with MSD and SIM - total ion chromatogram from SIM (A) and extracted ion chromatogram from SIM(B)

Copyright 2000 Agilent Technologies Released 09/2000 Publication Number 5980-2499EN