ANTImiCROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1977, Copyright © 1977 American Society for Microbiology
p.
651-55
Vol. 11, No. 4 Printed in U.S.A.
Assay of Gentamicin in Serum by High-Pressure Liquid Chromatography JOHN P. ANHALT Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901 Received for publication 26 November 1976
A procedure is described that uses high-pressure liquid chNmatography for of gentamicin in serum. The technique involves extraction of gentamicin from serum by using a CM-Sephadex column and analysis by reverse-phase, ion-pair chromatography. Continuous-flow, post-column derivatization with o-phthaladehyde is used to form fluorescent products for detection. The possible scope of this method for analysis of other aminoglycoside antibiotics is illustrated by application to sisomicin and netilmicin. assay
o-Phthalaldehyde reagent (OPA). Potassium borate buffer was prepared by the titration of boric acid (24.7 g, 0.4 mol) dissolved in 900 ml of water with concentrated potassium hydroxide solution to pH 10.40, followed by dilution to 1.0 liter. OPA was prepared by dissolving o-phthalaldehyde (60 mg) in 1 ml of methanol, followed by addition of 2-mercaptoethanol (0.2 ml). The solution was gently mixed until complete decolorization occurred. Potassium borate buffer (100 ml) was then added with vigorous stirring. The solution was placed in the supply vessel for OPA, flushed several times with nitrogen, and used within 2 days. Antibiotic solutions. All antibiotic concentrations were calculated on the basis of activity. Stock solutions containing 1,000 ,ug of antibiotic per ml were prepared in 0.1 M phosphate buffer, pH 8.0, and stored at -20°C. Appropriate dilutions of the stock solutions were made with water. A serum solution containing 10 ug of gentamicin per ml was prepared by addition of an appropriate amount of aqueous standard containing 100 ,ug/ml of serum. Additional dilutions of this solution in serum were made to obtain other concentrations. Apparatus. The chromatographic equipment was constructed from commercially available components. A Tracor model 990 pump (Tracor Instruments, Austin, Tex.) was used to deliver mobile MATERIALS AND METHODS phase. A Schoeffel model 970 fluorometer (Schoeffel The purified gentamicin components C,, C,, and Instrument Corp., Westwood, N.J.) was used to deCl were kindly provided by J. Allan Waitz (Schering tect fluorescent products formed by continuous-flow, Corp., Bloomfield, N.J.). Gentamicin sulfate (USP post-column derivatization with OPA. Fluorescence reference standard), sisomicin sulfate, and netilmi- excitation was at 340 nm, and a KV418 filter was cin (Sch 20569) were also obtained from Schering used for emission. Photomultiplier voltage was varCorp. o-Phthalaldehyde (Fluoropa, Durrum) was ied between 1,000 and 1,100 V, depending on the obtained from Pierce Chemical Co., Rockford, Il. sensitivity required. OPA was supplied to a mixing The 2-mercaptoethanol was from Sigma Chemical tee from a pressurized glass vessel. A Cheminert Co., St. Louis, Mo. Methanol was obtained from fitting (CJ3031, Laboratory Data Control, Riviera Burdick and Jackson Laboratories, Muskegon, Beach, Fla.) was used for the mixing tee, and a Mich. Sodium pentanesulfonate was obtained from delay coil consisting of a Teflon tube (2.0 m by 0.6 Eastman Kodak Co., Rochester, N.Y. CM-Sephadex mm ID) was used between the mixing tee and detec(C-25) was from Pharmacia Fine Chemicals, Inc., tor. Analysis was performed by using a ,u-Bondapak Piscataway, N.J. Human serum was obtained from C18 column (30 cm by 3.9 mm ID) (Water Associates, a healthy volunteer. Water was deionized and glass Milford, Mass.) with a precolumn (4.3 cm by 4.2 mm ID) packed with Micropart C18 phase-bonded silica distilled. All other chemicals were of reagent grade. 651
High-pressure liquid chromatography is well suited for the analysis of polar, water-soluble compounds. These characteristics are common to many antibiotics, and procedures for analysis of some of these in serum have been developed (2, 3, 6). To attain the necessary sensitivity, detection was based upon ultraviolet absorption in each ofthese procedures. Aminoglycoside antibiotics, however, present a special problem in that they are not easily detected by this method. One approach to this problem would be to derivatize the antibiotic to give a more easily detectable compound. Under alkaline conditions, o-phthalaldehyde reacts rapidly with primary amines to give fluorescent products (1, 7). Chromatographic analysis of kanamycin using continuous-flow, post-column derivatization with this reagent has been reported (5). This procedure, however, was not applied to determinations in serum. I wish to report the application of high-pressure liquid chromatography to analysis of gentamicin in serum.
652
ANTIMICROB. AGENTS CHEMOTHER.
ANHALT
gel (5 Am; Applied Science Laboratories, State College, Pa.). Detector signal was processed and recorded by using a CDS 101 computing integrator (Varian, Palo Alto, Calif.) and a model 7123A recorder (Hewlett-Packard Co., Avondale, Pa.). Samples were injected by using a Valco CV-6-VHPa-C20 injection valve with a 15-,ul injection loop modified for variable volume injection (Glenco Scientific, Houston, Tex). Chromatographic conditions. Mobile-phase compositions are expressed as the ratio of components, by volume, added to give the final solution. No correction was made for volume changes as a result of mixing. Predominantly aqueous mobile phases were degassed before use by vacuum filtration through a Millipore type HA (0.45 um) membrane filter (Millipore Corp., Bedford, Mass); solutions that were predominantly methanol were filtered through a solvent-resistant type FH (0.5 pm) filter. The mobile phase used for analyses contained 0.2 M Na2SO,, 0.02 M sodium pentanesulfonate, and 0.1% (vol/vol) acetic acid in a water-methanol (97:3) mixture. Column flow rate was 2 ml/min at 184 atm. OPA flow rate was about 0.5 ml/min. Serum preparation. Gentamicin was separated from interfering compounds in serum by ion-exchange gel chromatography. A short (1.5-cm) column with a bed volume of 1 ml was prepared from CM-Sephadex (C-25), using 0.2 M Na2SO4 as the initial buffer. Serum (400 ,ul) was applied to this column and then eluted in succession with 1 ml and 4 ml of initial buffer. The eluting buffer was changed to 0.01 M NaOH in 0.2 M Na2SO4, and 600 IAI of this buffer was added. After the column had drained completely, a second volume of alkaline buffer (400 ul) was added and the eluate was collected. This fraction was used for chromatographic analysis. Volume injected was 15 ,ul.
tamicin-free serum at the same sensitivity is, shown in Fig. 5. There are no peaks in this chromatogram that would interfere with the gentamicin components, sisomicin, or netilmicin. The detector sensitivity used for these determinations was approximately four times that used to obtain the chromatogram in Fig. 1, and long-term fluctuations in base line were probably due to variations in the OPA flow rate.
The recovery of gentamicin added to serum determined by comparing the component
was
(3
%N-j
I RESULTS I Analysis of aqueous standards. The chromatogram of an aqueous sample containing 2.5 I ,ug of each of the purified gentamicin compo4 8 0 nents per ml is shown in Fig. 1. Individual components were used to determine the order Time (min) and position of elution. The small peaks occurFIG. 1. Chromatogram of a mixture ofgentamicin ring before and after gentamicin C,, presumcomponents Cl, C2, and C1 (2.5 pg/ml in each comably represent minor components of the genta- ponent). Volume injected: 15 pd. Chart speed: 025 micin complex (Fred Sancilio, Schering Corp., inch/min. personal communication). Three irnections of this mixture were made to demonstrate reproTABLE 1. Comparison of peak heights for three ducibility (Table 1). Figures 2 and 3 show chroanalyses of a standard mixture of gentamicin matograms of sisomicin (10 ug/ml) and netilmicomponents cin (10 ,ug/ml), respectively. The apparent imPeak height purities evident in the chromatogram of netilmicin were present in repeat analyses of this Injection Ci cl. Cs material and are as yet unidentified. 1 1.00 1.00 1.00 Analysis of gentamicin in serum. A chro2 0.95 0.96 0.95 matogram of the gentamicin-containing frac3 0.94 0.97 0.93 tion from a serum sample containing 1.0 Mg of a The peak height for each component is given gentamicin per ml is shown in Fig. 4. For comparison, the chromatogram obtained from gen- relative to the first injection. I
I
I
GENTAMICIN ASSAY
VOL. 11, 1977
:2
Z
653
Q3
q
QZ
;ZZ .q
8
4
0 ._
Time ( min) FIG. 3. Chromatogram of netilmicin (10 pg/ml). Volume injected: 10 pi. Chart speed: 0.25 inch/min.
t)
4 0 Time (mi n) FIG. 2. Chromatogram of sisomicin (10 pg/ml). Volume injected: 10 pl. Chart speed: 025 inch/min.
K)
K) peak heights from a serum sample containing 10 ,ug of gentamicin per ml with the results Z. obtained from an aqueous standard of the same *q) concentration. These results (Table 2) indicate that recovery is greater than 95% and that no significant change in relative peak height occurs as a result of the extraction procedure. Serum samples containing 1.0, 2.0, 3.0, 5.0, and 10.0 ug of gentamicin per ml were prepared and analyzed in order to determine the standard curve shown in Fig. 6. For each concentration, duplicate injections were made, and the sums of the component peak heights were averaged. A linear least-squares regression analysis gave a coefficient of correlation (r) of 0.997. 4 8 0 DISCUSSION Time (min ) Thin-layer chromatography (10) and ion-exchange chromatography (8) have been used to FIG. 4. Chromatogram obtained from serum conseparate the gentamicin components; however, taining 1.0 pg of gentamicin per ml. Volume inreverse-phase chromatography using a C18 jected: 15 pi. Chart speed: 025 inch/min.
654
ANHALT
ANTIMICROB. AGENTS CHEMOTHER.
.A)
0
4 8 Time (min)
FIG. 5. Chromatogram obtained from gentamicin-free serum. Volume injected: 15 pi. Chart speed: 0.25 inch/min. TABLE 2. Recovery of gentamicin from serum Component Component
rumIwater)a
cia
1.04
C2
0.96 0.95
C1 Sum a
Peak height ratio (se-
0.99
Average of two determinations.
y=4.72 x+ 1.36 r = 0.997
1
2 3 4 5 6 7 8 Gentamicin concentration
9 10
(,Ug/ml ) FIG. 6. Standard curve for gentamicin in serum. Each point represents the average of two determinations.
phase-bonded microparticulate silica gel column was chosen for this study. This type of column usually provides rapid separations that are related to the nonpolar differences between similar polar molecules. In addition, retention of charged compounds can be affected by ionpair formation with a suitable reagent in the mobile phase (9). The separations demonstrated in this study, therefore, are probably due to ion-pair formation between protonated aminoglycoside molecules and pentanesulfonate anions, with differences in the interaction of nonpolar groups with the reverse-phase column accounting for the observed order of elution. The mobile-phase composition, particularly with regard to incorporation of 0.2 M Na2SO4, was critical. In the early phases of this study, chromatography was attempted with various sodium sulfonate ion-pair reagents, but without Na2SO4 added. These experiments resulted in skewed peaks, poor resolution of the gentamicin components, and mobile phases with high methanol concentrations. Increased concentration of ion-pair reagent in reverse-phase chromatography should result in increased retention time (9); however, a paradoxical decrease in retention time occurred. Since a polyvalent cation, such as gentamicin, would exist largely as undissociated ion pairs in a lowionic-strength mobile phase (4), this effect was probably related to increased dissociation of gentamicin ion pairs as a result of increased ionic strength. With 0.2 M Na2SO4 in the mobile phase, the expected increase in retention time with increased ion-pair reagent concentration was observed. The extraction experiments with serum (Table 2) indicate that almost quantitative recovery of gentamicin is possible by using a CMSephadex column. Since no dilution of specimen occurs in this procedure, a 2.5-fold concentration can be accomplished if 1.0 ml, instead of 400 ul, of serum is extracted (unpublished data). A standard curve (Fig. 6) shows linear response over at least a 10-fold range in concentration. The procedure used for this study, however, did not incorporate an internal standard. Therefore, results might have been affected by such variables as evaporation, nonspecific adsorption ofdrug to glass surfaces, and variation in the volume of eluate collected. LITERATURE CITED 1. Benson, J. R., and P. E. Hare. 1975. o-Phthalaldehyde: fluorogenic detection of primary amines in the picomole range. Comparison with fluorescamine and ninhydrin. Proc. Natl. Acad. Sci. U.S.A. 72:619-622. 2. Blair, A. D., A. W. Forrey, B. T. Meijsen, and R. E. Cutler. 1975. Assay of flucytosine and furosemide by
VOL. 11, 1977
3.
high-pressure liquid chromatography. J. Pharm. Sci. 64:1334-1339. Cooper, M. J., M. W. Anders, and B. L. Mirkin. 1973. Ion-pair extraction and high-speed liquid chromatography of cephalothin and deacetylcephalothin in human serum and urine. Drug Metab. Dispos. 1:659-
662. 4. Hine, J. 1962. Physical organic chemistry, p. 73-75. McGraw-Hill, New York. 5. Mays, D. L., R. J. Van Apeldoorn, and R. G. Lauback. 1976. High-performance liquid chromatographic determination of kanamycin. J. Chromatogr. 120:93102. 6. Nilsson-Ehle, I., T. T. Yoshikawa, M. C. Schotz, and L. B. Guze. 1976. Quantitation of antibiotics using
GENTAMICIN ASSAY 7.
8. 9. 10.
655
high-pressure liquid chromatography: tetracyline. Antimicrob. Agents Chemother. 9:754-760. Roth, M. 1971. Fluorescence reaction for amino acids. Anal. Chem. 43:880-882. Thomas, A. H., and S. D. Tappin. 1974. Separation of gentamicin complex by ion-exchange chromatography. J. Chromatogr. 97:280-283. Wahlund, K.-G. 1975. Reversed-phase ion-pair partition chromatography of carboxylates and sulfonates. J. Chromatogr. 115:411-422. Wilson, W. L., G. Richard, and D. W. Hughes. 1973. Chemical determination of component ratio and potency of gentamicin complex. J. Pharn. Sci. 62:282284.
p.
651-55
Vol. 11, No. 4 Printed in U.S.A.
Assay of Gentamicin in Serum by High-Pressure Liquid Chromatography JOHN P. ANHALT Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901 Received for publication 26 November 1976
A procedure is described that uses high-pressure liquid chNmatography for of gentamicin in serum. The technique involves extraction of gentamicin from serum by using a CM-Sephadex column and analysis by reverse-phase, ion-pair chromatography. Continuous-flow, post-column derivatization with o-phthaladehyde is used to form fluorescent products for detection. The possible scope of this method for analysis of other aminoglycoside antibiotics is illustrated by application to sisomicin and netilmicin. assay
o-Phthalaldehyde reagent (OPA). Potassium borate buffer was prepared by the titration of boric acid (24.7 g, 0.4 mol) dissolved in 900 ml of water with concentrated potassium hydroxide solution to pH 10.40, followed by dilution to 1.0 liter. OPA was prepared by dissolving o-phthalaldehyde (60 mg) in 1 ml of methanol, followed by addition of 2-mercaptoethanol (0.2 ml). The solution was gently mixed until complete decolorization occurred. Potassium borate buffer (100 ml) was then added with vigorous stirring. The solution was placed in the supply vessel for OPA, flushed several times with nitrogen, and used within 2 days. Antibiotic solutions. All antibiotic concentrations were calculated on the basis of activity. Stock solutions containing 1,000 ,ug of antibiotic per ml were prepared in 0.1 M phosphate buffer, pH 8.0, and stored at -20°C. Appropriate dilutions of the stock solutions were made with water. A serum solution containing 10 ug of gentamicin per ml was prepared by addition of an appropriate amount of aqueous standard containing 100 ,ug/ml of serum. Additional dilutions of this solution in serum were made to obtain other concentrations. Apparatus. The chromatographic equipment was constructed from commercially available components. A Tracor model 990 pump (Tracor Instruments, Austin, Tex.) was used to deliver mobile MATERIALS AND METHODS phase. A Schoeffel model 970 fluorometer (Schoeffel The purified gentamicin components C,, C,, and Instrument Corp., Westwood, N.J.) was used to deCl were kindly provided by J. Allan Waitz (Schering tect fluorescent products formed by continuous-flow, Corp., Bloomfield, N.J.). Gentamicin sulfate (USP post-column derivatization with OPA. Fluorescence reference standard), sisomicin sulfate, and netilmi- excitation was at 340 nm, and a KV418 filter was cin (Sch 20569) were also obtained from Schering used for emission. Photomultiplier voltage was varCorp. o-Phthalaldehyde (Fluoropa, Durrum) was ied between 1,000 and 1,100 V, depending on the obtained from Pierce Chemical Co., Rockford, Il. sensitivity required. OPA was supplied to a mixing The 2-mercaptoethanol was from Sigma Chemical tee from a pressurized glass vessel. A Cheminert Co., St. Louis, Mo. Methanol was obtained from fitting (CJ3031, Laboratory Data Control, Riviera Burdick and Jackson Laboratories, Muskegon, Beach, Fla.) was used for the mixing tee, and a Mich. Sodium pentanesulfonate was obtained from delay coil consisting of a Teflon tube (2.0 m by 0.6 Eastman Kodak Co., Rochester, N.Y. CM-Sephadex mm ID) was used between the mixing tee and detec(C-25) was from Pharmacia Fine Chemicals, Inc., tor. Analysis was performed by using a ,u-Bondapak Piscataway, N.J. Human serum was obtained from C18 column (30 cm by 3.9 mm ID) (Water Associates, a healthy volunteer. Water was deionized and glass Milford, Mass.) with a precolumn (4.3 cm by 4.2 mm ID) packed with Micropart C18 phase-bonded silica distilled. All other chemicals were of reagent grade. 651
High-pressure liquid chromatography is well suited for the analysis of polar, water-soluble compounds. These characteristics are common to many antibiotics, and procedures for analysis of some of these in serum have been developed (2, 3, 6). To attain the necessary sensitivity, detection was based upon ultraviolet absorption in each ofthese procedures. Aminoglycoside antibiotics, however, present a special problem in that they are not easily detected by this method. One approach to this problem would be to derivatize the antibiotic to give a more easily detectable compound. Under alkaline conditions, o-phthalaldehyde reacts rapidly with primary amines to give fluorescent products (1, 7). Chromatographic analysis of kanamycin using continuous-flow, post-column derivatization with this reagent has been reported (5). This procedure, however, was not applied to determinations in serum. I wish to report the application of high-pressure liquid chromatography to analysis of gentamicin in serum.
652
ANTIMICROB. AGENTS CHEMOTHER.
ANHALT
gel (5 Am; Applied Science Laboratories, State College, Pa.). Detector signal was processed and recorded by using a CDS 101 computing integrator (Varian, Palo Alto, Calif.) and a model 7123A recorder (Hewlett-Packard Co., Avondale, Pa.). Samples were injected by using a Valco CV-6-VHPa-C20 injection valve with a 15-,ul injection loop modified for variable volume injection (Glenco Scientific, Houston, Tex). Chromatographic conditions. Mobile-phase compositions are expressed as the ratio of components, by volume, added to give the final solution. No correction was made for volume changes as a result of mixing. Predominantly aqueous mobile phases were degassed before use by vacuum filtration through a Millipore type HA (0.45 um) membrane filter (Millipore Corp., Bedford, Mass); solutions that were predominantly methanol were filtered through a solvent-resistant type FH (0.5 pm) filter. The mobile phase used for analyses contained 0.2 M Na2SO,, 0.02 M sodium pentanesulfonate, and 0.1% (vol/vol) acetic acid in a water-methanol (97:3) mixture. Column flow rate was 2 ml/min at 184 atm. OPA flow rate was about 0.5 ml/min. Serum preparation. Gentamicin was separated from interfering compounds in serum by ion-exchange gel chromatography. A short (1.5-cm) column with a bed volume of 1 ml was prepared from CM-Sephadex (C-25), using 0.2 M Na2SO4 as the initial buffer. Serum (400 ,ul) was applied to this column and then eluted in succession with 1 ml and 4 ml of initial buffer. The eluting buffer was changed to 0.01 M NaOH in 0.2 M Na2SO4, and 600 IAI of this buffer was added. After the column had drained completely, a second volume of alkaline buffer (400 ul) was added and the eluate was collected. This fraction was used for chromatographic analysis. Volume injected was 15 ,ul.
tamicin-free serum at the same sensitivity is, shown in Fig. 5. There are no peaks in this chromatogram that would interfere with the gentamicin components, sisomicin, or netilmicin. The detector sensitivity used for these determinations was approximately four times that used to obtain the chromatogram in Fig. 1, and long-term fluctuations in base line were probably due to variations in the OPA flow rate.
The recovery of gentamicin added to serum determined by comparing the component
was
(3
%N-j
I RESULTS I Analysis of aqueous standards. The chromatogram of an aqueous sample containing 2.5 I ,ug of each of the purified gentamicin compo4 8 0 nents per ml is shown in Fig. 1. Individual components were used to determine the order Time (min) and position of elution. The small peaks occurFIG. 1. Chromatogram of a mixture ofgentamicin ring before and after gentamicin C,, presumcomponents Cl, C2, and C1 (2.5 pg/ml in each comably represent minor components of the genta- ponent). Volume injected: 15 pd. Chart speed: 025 micin complex (Fred Sancilio, Schering Corp., inch/min. personal communication). Three irnections of this mixture were made to demonstrate reproTABLE 1. Comparison of peak heights for three ducibility (Table 1). Figures 2 and 3 show chroanalyses of a standard mixture of gentamicin matograms of sisomicin (10 ug/ml) and netilmicomponents cin (10 ,ug/ml), respectively. The apparent imPeak height purities evident in the chromatogram of netilmicin were present in repeat analyses of this Injection Ci cl. Cs material and are as yet unidentified. 1 1.00 1.00 1.00 Analysis of gentamicin in serum. A chro2 0.95 0.96 0.95 matogram of the gentamicin-containing frac3 0.94 0.97 0.93 tion from a serum sample containing 1.0 Mg of a The peak height for each component is given gentamicin per ml is shown in Fig. 4. For comparison, the chromatogram obtained from gen- relative to the first injection. I
I
I
GENTAMICIN ASSAY
VOL. 11, 1977
:2
Z
653
Q3
q
QZ
;ZZ .q
8
4
0 ._
Time ( min) FIG. 3. Chromatogram of netilmicin (10 pg/ml). Volume injected: 10 pi. Chart speed: 0.25 inch/min.
t)
4 0 Time (mi n) FIG. 2. Chromatogram of sisomicin (10 pg/ml). Volume injected: 10 pl. Chart speed: 025 inch/min.
K)
K) peak heights from a serum sample containing 10 ,ug of gentamicin per ml with the results Z. obtained from an aqueous standard of the same *q) concentration. These results (Table 2) indicate that recovery is greater than 95% and that no significant change in relative peak height occurs as a result of the extraction procedure. Serum samples containing 1.0, 2.0, 3.0, 5.0, and 10.0 ug of gentamicin per ml were prepared and analyzed in order to determine the standard curve shown in Fig. 6. For each concentration, duplicate injections were made, and the sums of the component peak heights were averaged. A linear least-squares regression analysis gave a coefficient of correlation (r) of 0.997. 4 8 0 DISCUSSION Time (min ) Thin-layer chromatography (10) and ion-exchange chromatography (8) have been used to FIG. 4. Chromatogram obtained from serum conseparate the gentamicin components; however, taining 1.0 pg of gentamicin per ml. Volume inreverse-phase chromatography using a C18 jected: 15 pi. Chart speed: 025 inch/min.
654
ANHALT
ANTIMICROB. AGENTS CHEMOTHER.
.A)
0
4 8 Time (min)
FIG. 5. Chromatogram obtained from gentamicin-free serum. Volume injected: 15 pi. Chart speed: 0.25 inch/min. TABLE 2. Recovery of gentamicin from serum Component Component
rumIwater)a
cia
1.04
C2
0.96 0.95
C1 Sum a
Peak height ratio (se-
0.99
Average of two determinations.
y=4.72 x+ 1.36 r = 0.997
1
2 3 4 5 6 7 8 Gentamicin concentration
9 10
(,Ug/ml ) FIG. 6. Standard curve for gentamicin in serum. Each point represents the average of two determinations.
phase-bonded microparticulate silica gel column was chosen for this study. This type of column usually provides rapid separations that are related to the nonpolar differences between similar polar molecules. In addition, retention of charged compounds can be affected by ionpair formation with a suitable reagent in the mobile phase (9). The separations demonstrated in this study, therefore, are probably due to ion-pair formation between protonated aminoglycoside molecules and pentanesulfonate anions, with differences in the interaction of nonpolar groups with the reverse-phase column accounting for the observed order of elution. The mobile-phase composition, particularly with regard to incorporation of 0.2 M Na2SO4, was critical. In the early phases of this study, chromatography was attempted with various sodium sulfonate ion-pair reagents, but without Na2SO4 added. These experiments resulted in skewed peaks, poor resolution of the gentamicin components, and mobile phases with high methanol concentrations. Increased concentration of ion-pair reagent in reverse-phase chromatography should result in increased retention time (9); however, a paradoxical decrease in retention time occurred. Since a polyvalent cation, such as gentamicin, would exist largely as undissociated ion pairs in a lowionic-strength mobile phase (4), this effect was probably related to increased dissociation of gentamicin ion pairs as a result of increased ionic strength. With 0.2 M Na2SO4 in the mobile phase, the expected increase in retention time with increased ion-pair reagent concentration was observed. The extraction experiments with serum (Table 2) indicate that almost quantitative recovery of gentamicin is possible by using a CMSephadex column. Since no dilution of specimen occurs in this procedure, a 2.5-fold concentration can be accomplished if 1.0 ml, instead of 400 ul, of serum is extracted (unpublished data). A standard curve (Fig. 6) shows linear response over at least a 10-fold range in concentration. The procedure used for this study, however, did not incorporate an internal standard. Therefore, results might have been affected by such variables as evaporation, nonspecific adsorption ofdrug to glass surfaces, and variation in the volume of eluate collected. LITERATURE CITED 1. Benson, J. R., and P. E. Hare. 1975. o-Phthalaldehyde: fluorogenic detection of primary amines in the picomole range. Comparison with fluorescamine and ninhydrin. Proc. Natl. Acad. Sci. U.S.A. 72:619-622. 2. Blair, A. D., A. W. Forrey, B. T. Meijsen, and R. E. Cutler. 1975. Assay of flucytosine and furosemide by
VOL. 11, 1977
3.
high-pressure liquid chromatography. J. Pharm. Sci. 64:1334-1339. Cooper, M. J., M. W. Anders, and B. L. Mirkin. 1973. Ion-pair extraction and high-speed liquid chromatography of cephalothin and deacetylcephalothin in human serum and urine. Drug Metab. Dispos. 1:659-
662. 4. Hine, J. 1962. Physical organic chemistry, p. 73-75. McGraw-Hill, New York. 5. Mays, D. L., R. J. Van Apeldoorn, and R. G. Lauback. 1976. High-performance liquid chromatographic determination of kanamycin. J. Chromatogr. 120:93102. 6. Nilsson-Ehle, I., T. T. Yoshikawa, M. C. Schotz, and L. B. Guze. 1976. Quantitation of antibiotics using
GENTAMICIN ASSAY 7.
8. 9. 10.
655
high-pressure liquid chromatography: tetracyline. Antimicrob. Agents Chemother. 9:754-760. Roth, M. 1971. Fluorescence reaction for amino acids. Anal. Chem. 43:880-882. Thomas, A. H., and S. D. Tappin. 1974. Separation of gentamicin complex by ion-exchange chromatography. J. Chromatogr. 97:280-283. Wahlund, K.-G. 1975. Reversed-phase ion-pair partition chromatography of carboxylates and sulfonates. J. Chromatogr. 115:411-422. Wilson, W. L., G. Richard, and D. W. Hughes. 1973. Chemical determination of component ratio and potency of gentamicin complex. J. Pharn. Sci. 62:282284.
