ANALYTICAL
BIOCHEMISTRY
201,
99-102
(1992)
Microanalysis of ,&Cyclodextrin and Glucosyl-Pcyclodextrin in Human Plasma by High-Performance Liquid Chromatography with Pulsed Amperometric Detection’ Yoko Kubota, Masako Fukuda, Keiko Ohtsuji, and Kyoko Koizumi2 Faculty
Received
of
Pharmaceutical
August
Sciences, Mukogawa
Women’s University,
663, Japan
19, 1991
High-performance liquid chromatography with pulsed amperometric detection was applied to the determination of B-cyclodextrin (&CD) and glucosyl (G)-&CD in human plasma. They were well resolved from each other and from background components of plasma on a polymer-based reversed-phase column with 0.6% acetonitrile aqueous solution containing 1 mu sodium hydroxide as an eluent. The samples in the effluent were detected with a pulsed amperometric detector after postcolumn alkalization. The detection limits of &CD and G-&CD in plasma at a signal-to-noise ratio of 3 were 11 and 5 pmol, respectively. 0 1992 Academic Press,
Koshien Kyuban-cho, Nishirwmiya
Inc.
The Japanese government approved P-cyclodextrin (/3-CD)3 and a mixture of CDs as natural food additives and a-CD and #I-CD as drug additives for oral administration in 1982 and 1983, respectively. In recent years the number of papers and patents dealing with the practical applications of CDs has increased remarkably. The use of CD-containing drugs such as dinoprostone+ CD, alprostadil-o-CD, limaprost-o-CD (all from Ono Pharmaceutical, Japan), and benexate hydrochloride@-CD (Shionogi, Japan) in therapy was already approved in Japan. Also in Italy, piroxicam-&CD has been marketed since the beginning of 1989. In foods, ‘This work was supported in part by a subsidy from the Japan Shipbuilding Industry Foundation. ’ To whom correspondence should be addressed. 3 Abbreviations used: CD, cyclodextrin; G-P-CD, glucosyl-fl-cyciodextrin; HPLC, high-performance liquid chromatography; HPAEC, high-performance anion-exchange chromatography; PAD, pulsed amperometric detector. 0003-2697/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
CDs application has been widespread, e.g., for the preparation of powdered spices, flavors, tea, nuts, seeds, and vinegar; for the stabilization of flavors and fragrances; for the reduction of bitter tastes; and for deodorant effects. Thus many CDs have been ingested into the human body; nevertheless the distribution of CDs has been uncertain, because of the absence of a satisfactory method for microanalysis of CDs. We found previously that high-performance liquid chromatography (HPLC) on a reversed-phase column using a pump that minimized pulsating flow and a refractive index detector with high sensitivity was effective for microanalyses of P-CD in rat plasma (1). Frijlink et al. (2) reported the determination of CDs in biological fluids by HPLC with negative calorimetric detection using postcolumn complexation with phenolphthalein. However, these methods had the following drawbacks: an unstable baseline at high sensitivity and relatively high limit of detection. Recently, we applied high-performance anion exchange chromatography (HPARC) with pulsed amperometric detection, introduced by Rocklin and Pohl (3), to analysis of CDs and branched CDs and obtained excellent resolution and high sensitivity (4). The sensitivity of detection with a pulsed amperometric detector (PAD) is over one order of magnitude higher than those obtained by conventional detection methods and, in addition, the baseline stability is very good. In this work we examined HPLC for the microanalysis of @-CD and glucosyl (G)@-CD in human plasma, using PAD. MATERIALS
AND
METHODS
Materials
P-CD was a gift from Mercian (Fujisawa, Japan) and purified by recrystallization from hot water. G-&CD 99
Inc. reserved.
100
KUBOTA
ET AL.
was prepared and purified according to the reported method (5). All reagents were of analytical reagent grade. Water used for preparation of eluents was distilled, deionized, and purified (18 Ma cm) using a NANO-pure II (Barnstead, Newton, MA). Sodium hydroxide solutions used for preparation of eluent and for pH adjustment of effluent were prepared by dilution of carbonate-free 50% sodium hydroxide solution with purified water. The eluents were filtered through a 0.2-pm membrane filter. Human plasma was obtained from plural healthy human blood.
1 ‘1.; 100
nA
PCD
Apparatus
and Columns
HPLC was performed on a Dionex BioLC Model 4000i system equipped with a Model PAD 2 pulsed amperometric detector (both from Dionex, Sunnyvale, CA) consisting of an amperometric flow-through cell with a gold working electrode, a silver-silver chloride reference electrode, and a potentiostat. The columns used were a Dionex HPIC-AS6 (10 pm, 250 X 4:mm id.) equipped with an AG6 guard column (50 X 4-mm i.d.) (both from Dionex), a YMC-Pack A-312 ODS (5 pm, 150 X 6-mm id.) (YMC, Kyoto, Japan), an Asahipak GS-320 (9 pm, 500 X 7.6-mm i.d.), an Asahipak. ODP-50 (5 pm, 250 X 4.6-mm i.d.), and an Asahipak C8P-50 (5 pm, 250 X 4.6-mm i.d.) (all from Asahi Kasei, Tokyo, Japan). Chromatographic
Conditions
and Measurements
The following pulse potentials and durations were used for analysis of @CD and G-P-CD at range 2 (sampling period, 200 ms) as the optimal conditions: E, = 0.10 V (tl = 300 ms), E, = 0.60 V (tz = 120 ms), E3 = -0.80 V (5 = 300 ms). The response time of the PAD ‘2 dedector was set to 1.0 s. Eluents prepared daily were degassed by sonication under bubbling of helium gas and kept under a stream of helium. Separations on reversed-phase columns, YMC-Pack A-312 and Asahipak ODP-50, Asahipak CBP-50 were carried out at 30 and 5O”C, respectively, by using a TU-100 column oven (JASCO, Tokyo, Japan). Other separations were performed at ambient temperature. Nonalkaline or weakly alkaline effluents from reversed-phase columns were adjusted to pH over 13 with 1.5 M sodium hydroxide solution using an AMMS-1 anion micromembrane suppressor (Dionex) (6) prior to detection. Peak areas were calculated by the use of a Model 807-IT integrator (JASCO).
0-+
10
Retention
time
30 (mln)
FIG. 1. Chromatogram of B-CD standard (1 nmol) in plasma. Chromatographic conditions: column, Dionex HPIC-AS6 (250 X 4-mm i.d.); eluent, 150 mu sodium hydroxide solution containing 175 mu sodium acetate; flow rate, 1.0 ml/min; detector, PAD 2; temperature, ambient.
CD and G-B-CD were deproteinized by ultrafiltration using an MPS-1 micropartition system (Amicon, Lexington, MA). The membranes used were previously washed with water. A 50-~1 portion of the filtrate was loaded onto the column. RESULTS
Separation
AND
DISCUSSION
and Detection
Previously, (Y-, /3-, and r-CD and branched CDs were separated by HPAEC on an HPIC-AS6 column using 150 mM sodium hydroxide solution containing 200 mM sodium acetate as the eluent (4). Thus, analysis of B-CD in human plasma was first attempted by HPAEC using 150 mM sodium hydroxide solution containing 175 mM sodium acetate. However, elution of the background components of plasma interfered with the assay of B-CD at a lower concentration (Fig. 1). Then the method of Haginaka et al. (S), involving the separation on a reversed-phase column with acetonitorile-water as an eluent, effluent pH modification with a cation-exchange membrane reactor surrounded by 1.5 M sodium hydroxide solution, and detection with a PAD, was examined using a YMC-Pack A-312 column. Unfortunately this method was also unsuccessful, as a number of interfering peaks were observed around the Preparation of Plasma Samples peak of P-CD in chromatograms of plasma samples containing lower concentration (~1 nmol) of &CD. Several graded amounts of P-CD and a fixed amount Separations on Asahipak GS-320, Asahipak ODP-50, of G-P-CD as the internal standard or vice versa were dissolved in human plasma to prepare several kinds of and Asahipak CBP-50 columns were tried next. The first standard solutions. The plasma samples containing ,f3- column is packed with vinyl alcohol copolymer gel,
CHROMATOGRAPHY
!?! 0' 2
t 2.0
z u
t
al 2 m -$
OF CYCLODEXTRINS
1.0 I
t 1
0
I
1
I
0.2
I
0.4
I
I
1
0.6
0.6
Flow rate (ml/mid FIG. 2. Effect 0.3,0.4,0.5,0.6, tion amount of umn, Asahipak aqueous solution; dium hydroxide
of flow rate on PAD response. PAD responses at 0.2, and 0.7 ml/min, relative to that at 0.8 ml/min. InjecO-CD was 1 nmol. Chromatographic conditions: colCSP-50 (250 X 4.6-mm i.d.): eluent, 1% acetonitrile temperature, 50°C; postcolumn addition, 1.5 M sosolution at a flow rate of 1.0 mUmin.
which carries many uniformly distributed hydroxyl groups and can be used for HPLC in the dual mode of gel-permeation chromatography and adsorption chromatography. The exclusion limit (pullulan) is 40,000. The elution pattern of CDs and branched CDs with this column and water resembled that of the C&bonded silica and methanol-water, namely, the elution order of CDs was y-CD, (r-CD, and P-CD (7). It was advertised that plasma samples were injectable on this column
A
I
30
IN HUMAN
without pretreatment. The second and third columns are packed with C,,- and C,-bondedvinyl alcohol copolymer gel, respectively. All three columns have a wider pH range (2-13) for separation than that of silica-based columns (
BIOCHEMISTRY
201,
99-102
(1992)
Microanalysis of ,&Cyclodextrin and Glucosyl-Pcyclodextrin in Human Plasma by High-Performance Liquid Chromatography with Pulsed Amperometric Detection’ Yoko Kubota, Masako Fukuda, Keiko Ohtsuji, and Kyoko Koizumi2 Faculty
Received
of
Pharmaceutical
August
Sciences, Mukogawa
Women’s University,
663, Japan
19, 1991
High-performance liquid chromatography with pulsed amperometric detection was applied to the determination of B-cyclodextrin (&CD) and glucosyl (G)-&CD in human plasma. They were well resolved from each other and from background components of plasma on a polymer-based reversed-phase column with 0.6% acetonitrile aqueous solution containing 1 mu sodium hydroxide as an eluent. The samples in the effluent were detected with a pulsed amperometric detector after postcolumn alkalization. The detection limits of &CD and G-&CD in plasma at a signal-to-noise ratio of 3 were 11 and 5 pmol, respectively. 0 1992 Academic Press,
Koshien Kyuban-cho, Nishirwmiya
Inc.
The Japanese government approved P-cyclodextrin (/3-CD)3 and a mixture of CDs as natural food additives and a-CD and #I-CD as drug additives for oral administration in 1982 and 1983, respectively. In recent years the number of papers and patents dealing with the practical applications of CDs has increased remarkably. The use of CD-containing drugs such as dinoprostone+ CD, alprostadil-o-CD, limaprost-o-CD (all from Ono Pharmaceutical, Japan), and benexate hydrochloride@-CD (Shionogi, Japan) in therapy was already approved in Japan. Also in Italy, piroxicam-&CD has been marketed since the beginning of 1989. In foods, ‘This work was supported in part by a subsidy from the Japan Shipbuilding Industry Foundation. ’ To whom correspondence should be addressed. 3 Abbreviations used: CD, cyclodextrin; G-P-CD, glucosyl-fl-cyciodextrin; HPLC, high-performance liquid chromatography; HPAEC, high-performance anion-exchange chromatography; PAD, pulsed amperometric detector. 0003-2697/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
CDs application has been widespread, e.g., for the preparation of powdered spices, flavors, tea, nuts, seeds, and vinegar; for the stabilization of flavors and fragrances; for the reduction of bitter tastes; and for deodorant effects. Thus many CDs have been ingested into the human body; nevertheless the distribution of CDs has been uncertain, because of the absence of a satisfactory method for microanalysis of CDs. We found previously that high-performance liquid chromatography (HPLC) on a reversed-phase column using a pump that minimized pulsating flow and a refractive index detector with high sensitivity was effective for microanalyses of P-CD in rat plasma (1). Frijlink et al. (2) reported the determination of CDs in biological fluids by HPLC with negative calorimetric detection using postcolumn complexation with phenolphthalein. However, these methods had the following drawbacks: an unstable baseline at high sensitivity and relatively high limit of detection. Recently, we applied high-performance anion exchange chromatography (HPARC) with pulsed amperometric detection, introduced by Rocklin and Pohl (3), to analysis of CDs and branched CDs and obtained excellent resolution and high sensitivity (4). The sensitivity of detection with a pulsed amperometric detector (PAD) is over one order of magnitude higher than those obtained by conventional detection methods and, in addition, the baseline stability is very good. In this work we examined HPLC for the microanalysis of @-CD and glucosyl (G)@-CD in human plasma, using PAD. MATERIALS
AND
METHODS
Materials
P-CD was a gift from Mercian (Fujisawa, Japan) and purified by recrystallization from hot water. G-&CD 99
Inc. reserved.
100
KUBOTA
ET AL.
was prepared and purified according to the reported method (5). All reagents were of analytical reagent grade. Water used for preparation of eluents was distilled, deionized, and purified (18 Ma cm) using a NANO-pure II (Barnstead, Newton, MA). Sodium hydroxide solutions used for preparation of eluent and for pH adjustment of effluent were prepared by dilution of carbonate-free 50% sodium hydroxide solution with purified water. The eluents were filtered through a 0.2-pm membrane filter. Human plasma was obtained from plural healthy human blood.
1 ‘1.; 100
nA
PCD
Apparatus
and Columns
HPLC was performed on a Dionex BioLC Model 4000i system equipped with a Model PAD 2 pulsed amperometric detector (both from Dionex, Sunnyvale, CA) consisting of an amperometric flow-through cell with a gold working electrode, a silver-silver chloride reference electrode, and a potentiostat. The columns used were a Dionex HPIC-AS6 (10 pm, 250 X 4:mm id.) equipped with an AG6 guard column (50 X 4-mm i.d.) (both from Dionex), a YMC-Pack A-312 ODS (5 pm, 150 X 6-mm id.) (YMC, Kyoto, Japan), an Asahipak GS-320 (9 pm, 500 X 7.6-mm i.d.), an Asahipak. ODP-50 (5 pm, 250 X 4.6-mm i.d.), and an Asahipak C8P-50 (5 pm, 250 X 4.6-mm i.d.) (all from Asahi Kasei, Tokyo, Japan). Chromatographic
Conditions
and Measurements
The following pulse potentials and durations were used for analysis of @CD and G-P-CD at range 2 (sampling period, 200 ms) as the optimal conditions: E, = 0.10 V (tl = 300 ms), E, = 0.60 V (tz = 120 ms), E3 = -0.80 V (5 = 300 ms). The response time of the PAD ‘2 dedector was set to 1.0 s. Eluents prepared daily were degassed by sonication under bubbling of helium gas and kept under a stream of helium. Separations on reversed-phase columns, YMC-Pack A-312 and Asahipak ODP-50, Asahipak CBP-50 were carried out at 30 and 5O”C, respectively, by using a TU-100 column oven (JASCO, Tokyo, Japan). Other separations were performed at ambient temperature. Nonalkaline or weakly alkaline effluents from reversed-phase columns were adjusted to pH over 13 with 1.5 M sodium hydroxide solution using an AMMS-1 anion micromembrane suppressor (Dionex) (6) prior to detection. Peak areas were calculated by the use of a Model 807-IT integrator (JASCO).
0-+
10
Retention
time
30 (mln)
FIG. 1. Chromatogram of B-CD standard (1 nmol) in plasma. Chromatographic conditions: column, Dionex HPIC-AS6 (250 X 4-mm i.d.); eluent, 150 mu sodium hydroxide solution containing 175 mu sodium acetate; flow rate, 1.0 ml/min; detector, PAD 2; temperature, ambient.
CD and G-B-CD were deproteinized by ultrafiltration using an MPS-1 micropartition system (Amicon, Lexington, MA). The membranes used were previously washed with water. A 50-~1 portion of the filtrate was loaded onto the column. RESULTS
Separation
AND
DISCUSSION
and Detection
Previously, (Y-, /3-, and r-CD and branched CDs were separated by HPAEC on an HPIC-AS6 column using 150 mM sodium hydroxide solution containing 200 mM sodium acetate as the eluent (4). Thus, analysis of B-CD in human plasma was first attempted by HPAEC using 150 mM sodium hydroxide solution containing 175 mM sodium acetate. However, elution of the background components of plasma interfered with the assay of B-CD at a lower concentration (Fig. 1). Then the method of Haginaka et al. (S), involving the separation on a reversed-phase column with acetonitorile-water as an eluent, effluent pH modification with a cation-exchange membrane reactor surrounded by 1.5 M sodium hydroxide solution, and detection with a PAD, was examined using a YMC-Pack A-312 column. Unfortunately this method was also unsuccessful, as a number of interfering peaks were observed around the Preparation of Plasma Samples peak of P-CD in chromatograms of plasma samples containing lower concentration (~1 nmol) of &CD. Several graded amounts of P-CD and a fixed amount Separations on Asahipak GS-320, Asahipak ODP-50, of G-P-CD as the internal standard or vice versa were dissolved in human plasma to prepare several kinds of and Asahipak CBP-50 columns were tried next. The first standard solutions. The plasma samples containing ,f3- column is packed with vinyl alcohol copolymer gel,
CHROMATOGRAPHY
!?! 0' 2
t 2.0
z u
t
al 2 m -$
OF CYCLODEXTRINS
1.0 I
t 1
0
I
1
I
0.2
I
0.4
I
I
1
0.6
0.6
Flow rate (ml/mid FIG. 2. Effect 0.3,0.4,0.5,0.6, tion amount of umn, Asahipak aqueous solution; dium hydroxide
of flow rate on PAD response. PAD responses at 0.2, and 0.7 ml/min, relative to that at 0.8 ml/min. InjecO-CD was 1 nmol. Chromatographic conditions: colCSP-50 (250 X 4.6-mm i.d.): eluent, 1% acetonitrile temperature, 50°C; postcolumn addition, 1.5 M sosolution at a flow rate of 1.0 mUmin.
which carries many uniformly distributed hydroxyl groups and can be used for HPLC in the dual mode of gel-permeation chromatography and adsorption chromatography. The exclusion limit (pullulan) is 40,000. The elution pattern of CDs and branched CDs with this column and water resembled that of the C&bonded silica and methanol-water, namely, the elution order of CDs was y-CD, (r-CD, and P-CD (7). It was advertised that plasma samples were injectable on this column
A
I
30
IN HUMAN
without pretreatment. The second and third columns are packed with C,,- and C,-bondedvinyl alcohol copolymer gel, respectively. All three columns have a wider pH range (2-13) for separation than that of silica-based columns (
