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Combined liquid chromatography-mass spectrometry for trace analysis of pharmaceuticals
Nuclear Instruments and Methods 198 (1982) 165-167 North-Holland Publishing Company
165
COMBINED LIQUID C H R O M A T O G R A P H Y - M A S S S P E C T R O M E T R Y FOR TRACE ANALYSIS O F PHARMACEUTICALS Lothar SCHMIDT, Harald D A N I G E L , and Hartmut J U N G C L A S Kernchemie, Fachbereich Physikalische Chemie, Philipps-Universti~t, D-3550 Marburg, FRG
A 252Cf-plasma desorption mass spectrometer (PDMS) for the analysis of thin layers from nonvolatile organic samples has been set up to be combined with a liquid chromatograph. A novel interface performs the direct inlet of the liquid sample through a capillary into the vacuum system of the spectrometer. Samples of drugs are periodically collected, transferred to the ion source and analysed using a rotating disk. This on-line sample preparation has been tested for three antiarrhythmic drugs using various solvents and mixtures.
1. Introduction The detection of pharmaceuticals in biological fluids like blood and urine requires a careful sample preparation and a sensitive detection system. In this sense high-performance liquid chromatography (HPLC) is a very sensitive analytical method [1]. Utilizing the proper detection for the desired substance, concentrations like 1 n g / m l and lower can be determined. In general mass spectrometry (MS) is very sensitive too but also produces specific information on many substances, however, depending on the ionization mode employed. A combination of these two methods ( L C / M S ) should be very powerful - similar to G C / M S - independent if one calls MS an universal detector for LC or calls LC just a sample clean-up for MS. Some commercially available L C / M S systems utilize the moving belt interface [2] in combination with electron impact and chemical ionization (El, CI). Since HPLC is suitable for nonvolatile organic samples, it is desirable to combine HPLC and a surface ionization mode with desorption, induced by fast atoms (SIMS), laser pulses (LIMS) or fission fragments (252Cf-PDMS). These three methods produce similar mass spectroscopical information for thermic unstable and non-volatile molecules [3]. Applying 252Cf-PDMS [4] we developed a novel L C / M S interface [5] which couples the LC on-line to the MS using a capillary inlet. A vacuum lock
allows also the off-line analysis of already prepared samples. For some antiarrhythmic drugs we investigated the detection limits and the influence of solvents on the relative detection efficiency.
2. Equipment The system, which was set-up with the intention to combine 2~2Cf-PDMS [4] and reversed-phase LC was described recently [5]. Section 2.1 shall explain further modifications. 2.1. The time-of-flight mass spectrometer
The present set-up is schematically shown in fig. l. The 252Cf source (1 #Ci) is mounted behind
e- -CONVERTER (-15 kV)
SAMPLE
GRIDS
(-+10 kV)
ON GROUND
e--CONVERTER
/ /,
( - t 5 kV)
I
n15keV
I
MCP
~)'
*-10keYIONS 252Cf
START STOP• TIMINGSIGNALS
11,
MCP
x~ ~
CONDENSER
Fig. 1: Schematic drawing of the 252Cf-PDMS time-of-flight spectrometer displaying the ion source, flight path and ion detection via conversion electrons (MCP: micro channel plates).
0167-5087/82/0000-0000/$02.75 © 1982 North-Holland
IV. APPLICATIONS
L. Schmidt et al. / Trace analysis of pharmaceuticals
166
the sample foil. When a fission fragment passes this foil (100/s), the antiparallel fragment generates secondary electrons from a converter foil, which are detected by a micro channel plate (MCP) arrangement and finally produce a "start" timing signal. Also the desorbed ions ( ± 1 0 keV) are converted to electrons at the end of the drift tube (72 cm length), which are detected by a second MCP arrangement and produce the "stop" timing signals. Since the grid in front of the converter has 80% transmission, the detection efficiency for the ions arriving at the end of the flight path is ca. 80%. This improvement will be very useful, when a time digitizer with multi stop logic (buffer memory) is utilized instead of a conventional time to amplitude converter.
2.2. The L C / M S interface This novel interface combines LC and 252CfPDMS in a two fold way: on-line by introducing the effluent continuously through a capillary inlet and off-line by transferrring prepared samples though a vacuum lock. The interface is a rotating disk containing 8 discrete sample positions and controlled by a stepping motor. Fig. 2 shows a cut through this disk displaying the capillary and the ion source positions. The disk is centered in the rough vacuum chamber and extends into the high vacuum (10 6 mbar) of the MS. Running a 500 1/s Roots pump the rough vacuum (10 2 mbar) is maintained even at 300 /~l/min solvent flow through the capillary inlet.
Due to the rapid decompression of the effluent, it can freeze at the tip of the capillary, especially when it contains water. To prevent plugging of the capillary its tip can be heated by a coil (ca. l0 W).
3. Experiments The system was tested with the pure antiarrhythmic drugs Bunitrolol.HC1, Quinidine.SO4, and Verapamil.HC1. The nominal molecular weights M of the free bases are 248, 324 and 454, which are observed in the mass spectra as (M + H) + peaks due to proton transfer. Pure samples and mixtures of these three drugs were desolved in various solvents, which are suitable for reversedphase LC: H20, CH3OH, CH3CN, and various mixtures of these. Mass spectra were recorded with acquisition times between 10 rain and 5 h, depending on the count rate. The intensities for the three molecular lines are deduced from background corrected peak areas and normalized to 100 min.
4. Results Usually samples for 252Cf-PDMS are prepared in advance by the electro-spray technique [6] and then transferred into the MS (off-line). Comparing the results for the pure pharmaceuticals (Bunitrolol, Quinidine and Verapamil) no significant difference in the count rates was observed in off-line and on-line samples preparation. But we found a large variation in molecular ion intensities for the three pharmaceuticals during on-line test experiments. The collection process of the pharmaceuti-
~J//'///////////////~ ................ t~'~ ROOTS• PUMP
!
HEATER~
I
~
104-
=~:~=====a=,~/~ CAPILLARY ~
A
FROM HPLC
A
E
O
oE 103 2
"~ ~2'I I~ ~0- mbor V/. n
~ii u,,,P
o 102'
[
[~'I
10-6mb°r -~ 10keY [0NS
I
,
o8 O Verapomtl
/'
............ ('v
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII]IIIIIIIIIIA
Fig. 2: L C / M S source.
6
z
~."/2~///////////~
O
interface, cut through capillary inlet and ion
A Quinidine 101
i
I0
i
102
i
103
i
i
104
10Sng/ml
Drug concentrotion
Fig. 3: M o l e c u l a r ion intensity as a function of drug concentration using a C H a O H : H 2 0 = 1 : 1 solvent.
L. Schmidt et al. / Trace analysis of pharmaceuticals
• 1000-
•
8unitrotol ~100 ng Verapamil I ~]-
167
Drugs (100 ng/ml)
1000-
_= 800 E
__. E
"~ 6ooz o
~ 1,00-
200" 200"
J
J
i 3
I0
102
10 ~g/ml
Sort concentration [(NHz.)H 2 POt,]
/
Salts ( 500 gig/roll [Z] NoCI I
[NH~) HzPO~
800-
600-
,ooi
- [-
5 I Bunitrolot
Quinidine
Verapamil
Fig. 4: Molecular ion intensity as a function of ( N H 4 ) H 2 P O 4 concentration, added to the C H 3 C N : H 2 0 = 4:1 solvent.
Fig. 5: Molecular ion intensity using a C H 3 C N : H 2 0 = 9:1 solvent and adding two different salts. The error bars indicate the standard deviation from 6 experiments per data point.
cals in the rough vacuum on the sample foil is very critical. If the pressure is too high, the sample molecules do not even stick to the foil. Also we observed that an already collected deposit was washed away by the following liquid jet. Fig. 3 shows the intensities for Quinidine and Verapamil as a function of drug concentration using a C H a O H : H 2 0 = 1:1 solvent. Using this solvent Bunitrolol was hardly detectable, but it worked very well with C H 3 C N - H 2 0 mixtures. We investigated the influence of (NH4)H=PO 4 added to the solvent. Fig. 4 displays the positive effect on Bunitrolol, while Verapamil is slowly varying and Quinidine hardly detectable. Since the effect is not uniform we used two different salts and repeated each experiment six times. Fig. 5 compares the influence of NaC1 and ( N H 4 ) H 2 P O 4. While Bunitrolol is enhanced by ( N H 4 ) H 2PO 4, Quinidine and Verapamil are more intense using NaCI.
using the capillary inlet is still in an empirical state. The collection efficiency of the sample molecules in a rough vacuum chamber, i.e. the sensitivity of the intented L C / M S analysis varies from pharmaceutical to pharmaceutical and depends on the solvents used. The efficiency can be improved by adding to the solvent salts, which act as condensation and crystallization centers.
5. Conclusion
The preparation of a sample from small amounts (<~ l0 ng in 100 /~1 solvent) in the on-line mode
This work was supported by the BMFT, Bonn.
References [1] G L C and HPLC Determination of Therapeutic Agents, Chromatographic Science Series, vol. 9, part 3, ed., K. Tsuji (Marcel Dekker, New York, 1979). [2] W.H. McFadden and D.C. Bradford, 25th Conf. Mass. Spectrom. Allied Topics, Paper FP-I1, Washington DC, May (1977). [3] Proc. of the Workshop Ion Formation from Organic Solids, Miinster (October 6-8, 1980) to be published by Springer. [4] R.D. Macfarlane and D.F. Torgerson, Int. J. Mass Spectrom. Ion. Phys. 21 (1976) 81. [5] H. Jungclas, H. Danigel, and L. Schmidt, Org. Mass Spectrom. 17 (1982) 86. [6] C.J. McNeal, R.D. Macfarlane, and E.L. Thurston, Anal. Chem. 51 (1979) 2036.
IV. A P P L I C A T I O N S
165
COMBINED LIQUID C H R O M A T O G R A P H Y - M A S S S P E C T R O M E T R Y FOR TRACE ANALYSIS O F PHARMACEUTICALS Lothar SCHMIDT, Harald D A N I G E L , and Hartmut J U N G C L A S Kernchemie, Fachbereich Physikalische Chemie, Philipps-Universti~t, D-3550 Marburg, FRG
A 252Cf-plasma desorption mass spectrometer (PDMS) for the analysis of thin layers from nonvolatile organic samples has been set up to be combined with a liquid chromatograph. A novel interface performs the direct inlet of the liquid sample through a capillary into the vacuum system of the spectrometer. Samples of drugs are periodically collected, transferred to the ion source and analysed using a rotating disk. This on-line sample preparation has been tested for three antiarrhythmic drugs using various solvents and mixtures.
1. Introduction The detection of pharmaceuticals in biological fluids like blood and urine requires a careful sample preparation and a sensitive detection system. In this sense high-performance liquid chromatography (HPLC) is a very sensitive analytical method [1]. Utilizing the proper detection for the desired substance, concentrations like 1 n g / m l and lower can be determined. In general mass spectrometry (MS) is very sensitive too but also produces specific information on many substances, however, depending on the ionization mode employed. A combination of these two methods ( L C / M S ) should be very powerful - similar to G C / M S - independent if one calls MS an universal detector for LC or calls LC just a sample clean-up for MS. Some commercially available L C / M S systems utilize the moving belt interface [2] in combination with electron impact and chemical ionization (El, CI). Since HPLC is suitable for nonvolatile organic samples, it is desirable to combine HPLC and a surface ionization mode with desorption, induced by fast atoms (SIMS), laser pulses (LIMS) or fission fragments (252Cf-PDMS). These three methods produce similar mass spectroscopical information for thermic unstable and non-volatile molecules [3]. Applying 252Cf-PDMS [4] we developed a novel L C / M S interface [5] which couples the LC on-line to the MS using a capillary inlet. A vacuum lock
allows also the off-line analysis of already prepared samples. For some antiarrhythmic drugs we investigated the detection limits and the influence of solvents on the relative detection efficiency.
2. Equipment The system, which was set-up with the intention to combine 2~2Cf-PDMS [4] and reversed-phase LC was described recently [5]. Section 2.1 shall explain further modifications. 2.1. The time-of-flight mass spectrometer
The present set-up is schematically shown in fig. l. The 252Cf source (1 #Ci) is mounted behind
e- -CONVERTER (-15 kV)
SAMPLE
GRIDS
(-+10 kV)
ON GROUND
e--CONVERTER
/ /,
( - t 5 kV)
I
n15keV
I
MCP
~)'
*-10keYIONS 252Cf
START STOP• TIMINGSIGNALS
11,
MCP
x~ ~
CONDENSER
Fig. 1: Schematic drawing of the 252Cf-PDMS time-of-flight spectrometer displaying the ion source, flight path and ion detection via conversion electrons (MCP: micro channel plates).
0167-5087/82/0000-0000/$02.75 © 1982 North-Holland
IV. APPLICATIONS
L. Schmidt et al. / Trace analysis of pharmaceuticals
166
the sample foil. When a fission fragment passes this foil (100/s), the antiparallel fragment generates secondary electrons from a converter foil, which are detected by a micro channel plate (MCP) arrangement and finally produce a "start" timing signal. Also the desorbed ions ( ± 1 0 keV) are converted to electrons at the end of the drift tube (72 cm length), which are detected by a second MCP arrangement and produce the "stop" timing signals. Since the grid in front of the converter has 80% transmission, the detection efficiency for the ions arriving at the end of the flight path is ca. 80%. This improvement will be very useful, when a time digitizer with multi stop logic (buffer memory) is utilized instead of a conventional time to amplitude converter.
2.2. The L C / M S interface This novel interface combines LC and 252CfPDMS in a two fold way: on-line by introducing the effluent continuously through a capillary inlet and off-line by transferrring prepared samples though a vacuum lock. The interface is a rotating disk containing 8 discrete sample positions and controlled by a stepping motor. Fig. 2 shows a cut through this disk displaying the capillary and the ion source positions. The disk is centered in the rough vacuum chamber and extends into the high vacuum (10 6 mbar) of the MS. Running a 500 1/s Roots pump the rough vacuum (10 2 mbar) is maintained even at 300 /~l/min solvent flow through the capillary inlet.
Due to the rapid decompression of the effluent, it can freeze at the tip of the capillary, especially when it contains water. To prevent plugging of the capillary its tip can be heated by a coil (ca. l0 W).
3. Experiments The system was tested with the pure antiarrhythmic drugs Bunitrolol.HC1, Quinidine.SO4, and Verapamil.HC1. The nominal molecular weights M of the free bases are 248, 324 and 454, which are observed in the mass spectra as (M + H) + peaks due to proton transfer. Pure samples and mixtures of these three drugs were desolved in various solvents, which are suitable for reversedphase LC: H20, CH3OH, CH3CN, and various mixtures of these. Mass spectra were recorded with acquisition times between 10 rain and 5 h, depending on the count rate. The intensities for the three molecular lines are deduced from background corrected peak areas and normalized to 100 min.
4. Results Usually samples for 252Cf-PDMS are prepared in advance by the electro-spray technique [6] and then transferred into the MS (off-line). Comparing the results for the pure pharmaceuticals (Bunitrolol, Quinidine and Verapamil) no significant difference in the count rates was observed in off-line and on-line samples preparation. But we found a large variation in molecular ion intensities for the three pharmaceuticals during on-line test experiments. The collection process of the pharmaceuti-
~J//'///////////////~ ................ t~'~ ROOTS• PUMP
!
HEATER~
I
~
104-
=~:~=====a=,~/~ CAPILLARY ~
A
FROM HPLC
A
E
O
oE 103 2
"~ ~2'I I~ ~0- mbor V/. n
~ii u,,,P
o 102'
[
[~'I
10-6mb°r -~ 10keY [0NS
I
,
o8 O Verapomtl
/'
............ ('v
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII]IIIIIIIIIIA
Fig. 2: L C / M S source.
6
z
~."/2~///////////~
O
interface, cut through capillary inlet and ion
A Quinidine 101
i
I0
i
102
i
103
i
i
104
10Sng/ml
Drug concentrotion
Fig. 3: M o l e c u l a r ion intensity as a function of drug concentration using a C H a O H : H 2 0 = 1 : 1 solvent.
L. Schmidt et al. / Trace analysis of pharmaceuticals
• 1000-
•
8unitrotol ~100 ng Verapamil I ~]-
167
Drugs (100 ng/ml)
1000-
_= 800 E
__. E
"~ 6ooz o
~ 1,00-
200" 200"
J
J
i 3
I0
102
10 ~g/ml
Sort concentration [(NHz.)H 2 POt,]
/
Salts ( 500 gig/roll [Z] NoCI I
[NH~) HzPO~
800-
600-
,ooi
- [-
5 I Bunitrolot
Quinidine
Verapamil
Fig. 4: Molecular ion intensity as a function of ( N H 4 ) H 2 P O 4 concentration, added to the C H 3 C N : H 2 0 = 4:1 solvent.
Fig. 5: Molecular ion intensity using a C H 3 C N : H 2 0 = 9:1 solvent and adding two different salts. The error bars indicate the standard deviation from 6 experiments per data point.
cals in the rough vacuum on the sample foil is very critical. If the pressure is too high, the sample molecules do not even stick to the foil. Also we observed that an already collected deposit was washed away by the following liquid jet. Fig. 3 shows the intensities for Quinidine and Verapamil as a function of drug concentration using a C H a O H : H 2 0 = 1:1 solvent. Using this solvent Bunitrolol was hardly detectable, but it worked very well with C H 3 C N - H 2 0 mixtures. We investigated the influence of (NH4)H=PO 4 added to the solvent. Fig. 4 displays the positive effect on Bunitrolol, while Verapamil is slowly varying and Quinidine hardly detectable. Since the effect is not uniform we used two different salts and repeated each experiment six times. Fig. 5 compares the influence of NaC1 and ( N H 4 ) H 2 P O 4. While Bunitrolol is enhanced by ( N H 4 ) H 2PO 4, Quinidine and Verapamil are more intense using NaCI.
using the capillary inlet is still in an empirical state. The collection efficiency of the sample molecules in a rough vacuum chamber, i.e. the sensitivity of the intented L C / M S analysis varies from pharmaceutical to pharmaceutical and depends on the solvents used. The efficiency can be improved by adding to the solvent salts, which act as condensation and crystallization centers.
5. Conclusion
The preparation of a sample from small amounts (<~ l0 ng in 100 /~1 solvent) in the on-line mode
This work was supported by the BMFT, Bonn.
References [1] G L C and HPLC Determination of Therapeutic Agents, Chromatographic Science Series, vol. 9, part 3, ed., K. Tsuji (Marcel Dekker, New York, 1979). [2] W.H. McFadden and D.C. Bradford, 25th Conf. Mass. Spectrom. Allied Topics, Paper FP-I1, Washington DC, May (1977). [3] Proc. of the Workshop Ion Formation from Organic Solids, Miinster (October 6-8, 1980) to be published by Springer. [4] R.D. Macfarlane and D.F. Torgerson, Int. J. Mass Spectrom. Ion. Phys. 21 (1976) 81. [5] H. Jungclas, H. Danigel, and L. Schmidt, Org. Mass Spectrom. 17 (1982) 86. [6] C.J. McNeal, R.D. Macfarlane, and E.L. Thurston, Anal. Chem. 51 (1979) 2036.
IV. A P P L I C A T I O N S