Anal. Chem. 1984, 56, 491-497
CONCLUSION For the UV-visualization analysis of sodium valproate, an eluent containing 5 mM KC1 has been shown to provide chromatographic performance which is superior to what is obtained by using an eluent containing no added salt. Stability of retention time and peak area, freedom from interference by ionic sample components or sample solvent, compatibility with the internal standard method, and precision of analysis are all benefits of using the high salt eluent. The implications of these findings for paired-ion chromatography in general are intriguing. Similar effects of ionic strength may be present for the usual paired-ion systems but might not ordinarily be noticed because the ion-pairing reagents are detector-transparent. Further research in this area is ongoing in our laboratory. ACKNOWLEDGMENT The authors wish to thank K. Bergeron and J. Newman for assistance in preparing the manuscript and P. Watson for preparation of the figures. LITERATURE CITED (1) Bidiingmeyer. B. A.; Deming, S. N.; Price, W. P.; Sachck, B.; Petrusek, M. J. Paper presented at Advances in Chromatography, 14th Interna-
49 1
tionai Symposium, Lausanne, Switzerland, Sept 24-28, 1979. Bidlingmeyer, 8. A,; Deming, S. N.; Price, W. P., Jr.; Sachok, B.; Petrusek, M. J . Chromatogr. 1079, 186, 419. Bdilngmeyer, B. A. J . Chromatogr. Scl. 1080, 78, 525. Bidiingmeyer, 6. A.; Deming, S. N.; Sachok, B. Poster presented at 13th International Symposium on Chromatography, Cannes, France, Julv 2. 1980. Sachok, B.; Deming. S. N.; Bdilngmeyer, B. A. J . Liq. Chromatogr. 1082.5.389. Paris,’ N.’ Anal. Blochem. 1070, 100. 250. Paris, N. J . Liq. Chromatogr. 3 , 1743. Helboe, P. J . Chromatogr. 1083, 261, 117. Denkert, M.; Hackzeii, L.; Schiii, G.; Sjogren, E. J . Chromatogr. 1081, 218, 31. Barber, W. E.; Carr, P. W. J . Chromatogr. 1083, 260, 89. Bidiingmeyer, B. A.; Warren, F. V., ;r. Anal. Chem. 1082, 54, 2351. Snyder, L. R.; Kirkiand, J. J. Introduction to Modern Liquid Chromatography”; Wiiey-Interscience: New York, 1974. Durst, H. D.; Miiano, M.; Kitka, E. J., Jr.; Connelly, S. A.; Grushka, E. Anal. Chem. 1075, 4 7 , 1797. Iskandarani, Z.; Pietrzyk, D. J. Anal. Chem. 1082, 5 4 , 1085. Stranahan, J. J.; Deming, S. N. Anal. Chem. 1882, 54, 1540.
RECEIVED for review January 25,1983. Resubmitted July 28, 1983. Accepted October 31,1983. Portions of this work were presented at the Fall 1982 ACS Meeting (KansasCity, MO), at the 1982 FACSS Meeting (Philadelphia, PA), and at the 1983 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Atlantic City, NJ).
Post-Column Reaction Detector for Platinum(I I) Antineoplastic Agents Kennan C. Marsh,l Larry A. Sternson,* and Arnold J. Repta
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66045
The development and evaluation of a post-column reaction detector sensitive to piathum( 11) complexes Is presented In which sodlum bisulfite Is used as the derivatlzlng agent wlth potassium dlchromate as an activating agent. The Influences of mobile phase changes (Le., pH, organic modlflers, electrolytes), oxygen, metal ions, and order of reagent addition on reactlon klnetlcs and product yield are defined and used 290 In optlmizatlon of detector response. Detectlon at A, nm results In an on-ilne post-column sensltivlty of 40-60 ng/mL for selected cls-dichloroplatinum complexes and a sensltlvity of 300-1200 ng/mL for four (substituted)malonato-piatlnum complexes. The reactlon detector Is used to monitor the klnetlcs of aquation ol clsplatln (CDDP) and to quantitate CDDP degradation In plasma. As the sensHlvity for CDDP in plasma is comparable to that achleved from HPLC effluent fractlonatlon/off-line flameless atomlc absorption (AAS) quantltatlon, slgnlficant utillty for this time-efflclent post-column reactor in cilnlcal analysis is suggested.
Currently the most widely accepted analytical procedure for quantitation of cisplatin (CDDP) (1-4) utilizes initial HPLC separation of the platinum species followed by off-line quantitation of the platinum in each fraction with flameless atomic absorption (AAS) (5-8). For time-efficient application in clinical analysis, the need for a selective direct on-line detector system was indicated. Recently, on-line electrochemical detection of HPLC effluent was shown to be useful Present address: Abbott Labs, D-493,North Chicago, IL 60064. 0003-2700/84/0356-0491$01.50/0
in the sensitive quantitation of both CDDP and CHIP (Table I) in urine samples but was not responsive to the other platinum antineoplastic agents investigated (9, 10). Spectrophotometric monitoring of column effluent is probably the preferred detection system in HPLC, but due to the poor molar extinction coefficients of the platinum(I1) compounds, a derivatization reaction was required to provide the sensitivity needed for clinical application. Nucleophiles which react rapidly with platinum(I1) would convert a variety of platinum compounds (differing in ligand composition) to a common final product (11). Therefore, to maintain the selectivity of the system, chromatographic separation of the underivatized compounds (12,13) must precede derivatization. In earlier studies of the reaction between CDDP and N,N’-bis (3-dimethylaminopropyl)dithiooxamide (DTO) (14 ) , a dramatic rate enhancing effect was observed when CDDP was exposed to (bi)sulfite prior to reacting with DTO. Hussain et al. (15) found that (bi)sulfite rapidly degrades cisplatin, resulting in product(s) exhibiting increased absorbance a t 290-300 nm. The rapid rate of reaction to form chromophoric product(s) indicated potential applicability of bisulfite as a derivatizing reagent in a post-column reactor. Variables affecting the CDDP/HS03- reaction were optimized and considered in the design of the post-column reactor. The response of the reactor is maximized, using CDDP as the model platinum compound, and then evaluated for a series of substituted platinum(I1) antineoplastic agents.
EXPERIMENTAL SECTION Apparatus. Spectrophotometric kinetic studies and spectral characterization of platinum compounds were obtained with a Perkin-Elmer (Norwalk,CT) 555 UV/vis spectrophotometer with 0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL.
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1984
Peltier system for temperature control. A Waters Associates (Milford, MA) M6000A solvent delivery system was used with a Rheodyne 7120 (Cotati, CA) fixed volume (20 pL) injector and a Waters 450 variable-wavelength detector for the HPLC based kinetic studies. Absorbance was monitored on a Houston Instruments (Houston, TX) Omniscribe single pen recorder, and peak areas were quantitated with a Varian (Sunnyvale, CA) CDS-111L integrator. Platinum analysis of HPLC fractions was obtained by using flameless atomic absorption (AAS) (Varian, Sunnyvale,CA) spectrometry according to the procedure described by Long (16). Reagents. Potassium hexachloroplatinate(1V) (Engelhard, Newark, NJ), potassium tetrachloroplatinate(I1) and cis-dichloro(ethylenediamine)platinum(II) (Alfa, Danvers, MA) were obtained in >99% purity. The other platinum-containing compounds (Table I) were obtained from the National Cancer Institute. Recovered human plasma was obtained from the Community Blood Center (Kansas City, MO). Water used throughout was deionized in mixed bed ion exchange columns followed by glass distillation. All other chemicals were of analytical grade and were used as received. Chromatographic Conditions. Analytical columns (100 x 4.6 mm and 150 X 4.6 mm, 5 pm ODS Hypersil (Shandon, Sewickly, PA)) were packed by using a high pressure upward slurry packing technique (17). HPLC studies of the kinetics of the cisplatin-bisulfite reaction utilized a solvent-generated anion exchange column (100 X 4.6 mm, ODS Hypersil, coated with hexadecyltrimethylammonium bromide (HTAB)) as described by Riley et al. (12) and an aqueous citrate buffer mobile phase (10 mM citrate, pH 5.25, lo4 M HTAB, 1.0 mL/min). The mobile phase pH was adjusted to 4.5 (10 mM citrate, low4M HTAB) to separate the cis-dichloroplatinumcomplexes (Pt(en)C12,DACC12, CDDP; see Table I) and maintained at pH 5.0 (10 mM citrate, M HTAB) for the quantitation of CDDP in plasma. The (substituted)malonato-platinumcompounds (DACMAL, CBCDA, MAL, MALOH) were separated on a 15-cm column (5 pm ODS Hypersil) by using acetate buffer (pH 4.5,lO mM) as the mobile phase. Kinetics of Bisulfite-Cisplatin Reaction in Solution: HPLC Studies. The instability of the bisulfite solution and the rapid rate of the CDDP/bisulfite reaction necessitated the use of a rigid timing sequence in the sample preparation. Two minutes after dissolutionof the NaHS03solid in diluent, an aliquot (-0.5 mL-volume calculated to give a final concentration = 2.52 X lod M) of CDDP stock solution (in 0.1 M NaCl) was added to the reaction flask (10-mL volumetric flask containing 9.3 mL of diluent). The appropriate aliquot (99.8-100.3 pL) of stock NaHS03 M NaHS03 in the solution (aliquot calculated to give 1.0 X reaction solution) was added to the reaction flask at 2 min, 35 s. The reaction solution was brought to volume (10 mL) with a few drops of diluent, mixed and an aliquot injected on the HPLC at 3 min, 35 s (1 min after reagent combination). Repetitive injections were made at time intervals sufficient to characterize the formation and decomposition of the major product at 300 nm (Figure 1). Kinetics were determined at ambient temperature (20 2 "C). In separate experiments, the diluent was either buffer, H20, or an aqueous solution of salts or metal ions. The influence of oxygen on the reaction was studied by alternately purging the bisulfite stock solution and/or the diluent (H20)with argon, oxygen, or air prior to initiation of the standard kinetic procedure. The effect of organic modifiers on the formation of the major reaction product was examined by adding 0.025 M DMF, dimethylacetamide (DMA), benzyl alcohol, methanol, or acetonitrile (ACN) to the diluent (H20)just prior to reagent addition. The effect of added salts on the reaction was studied in a series of separate experiments in which 0.1,0.5, 1.0, 5.0, and 10.0 mM solutionsof NaOAc, NaN03, NaC104,NaCl, citrate (pH 3.26,4.0), or phosphate (pH 6.88) were used as the diluent at constant concentrations of CDDP (2.52 X lod M) and NaHS03 (1.0 X M). To determine the effect of changing the HSO3-/S0;- ratio, the fraction of NaHS03 in stock solution was examined at 0.0, 0.25, 0.5,0.75, and 1.0 fraction increments with a total (bi)sulfite molarity of 0.1 M. Stock solutionsof ZnCl,, KMn04,CuS04,MnC12,and K2Cr207 (1 X lo-' M, 1 x lo4 M, 1 X M in distilled water) were used
*
L 1
I
0
5
t (rnin)
x lo4 M K2Cr207). The UV response to variable concentrations of CDDP (2.55 M, 0.077-12.0 pg/mL in 0.1 M NaC1) after X lo-' M to 4.0 X HPLC separation was determined before and after derivatization with bisulfite. The reaction detector response to other platinum M; Table I) was examined with antineoplastic agents (2.52 X reaction conditions optimized for CDDP. RESULTS AND DISCUSSION HPLC analysis of the CDDP/bisulfite reaction mixture resulted in the resolution of several species, with one major product predominating (Figure 1). The eluant fraction of each of these peaks was found to contain platinum (by AAS), but a majority (>60%) of the total platinum injected was found in the peak exhibiting maximum absorbance at 290 nm (Figure 1,peak 6). The bisulfite reaction with CDDP was too rapid, and the absorptivity of CDDP was too low to effectively monitor the loss of cisplatin chromatographically in the reaction mixture. However, the behavior of the major product (Figure 1, peak 6, k' = 4.4) appeared suitable for use in monitoring the reaction kinetics. Specific HPLC Kinetic Studies. Various components ordinarily present in HPLC chromatographic mobile phases (i.e., organic solvents, electrolytes, changes in pH) were in-
ANALYTICAL CHEMISTRY, VOL. 56,
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NO. 3, MARCH 1984 8 493
8
A 8 8
1 -
oe
-
06
0
40
80
I20
160
200
lime (minutes)
Flgure 2. Comparison of CDDP/NaHSO, reactions in which the diluent
was either purged with oxygen (A)or argon (m) or equilibrated to atmospheric conditions (0)prior to the reaction [condLions as in Figure 1, 300 nm] . vestigated to better assess their effect on the CDDP/HSOSreaction since the mobile phase represents the major reaction media component in a post-column reactor. The effects of changes in reagent concentration, added metal ions, and the presence of oxygen were also examined. Influence of Oxygen. Reactions in which both the bisulfite and reaction solutions were purged with argon exhibited slower rates of product formation and decomposition when compared to samples run under atmospheric conditions, while samples in which both solutions had been purged with oxygen exhibited more rapid rates of formation and decomposition (Figure 2). These observations indicated that 02 promotes not only the formation but also the decomposition of the chromophoric reaction product. Influence of Organic Modifiers. When equimolar concentrations (0.025 M) of DMF, DMA, benzyl alcohol, MeOH, or ACN were added to the reaction flask prior to the addition of bisulfite and cisplatin, each of the additives caused a significant decrease in the rate of product formation. If these organic modifiers (0.025 M) were added to the reaction mixture at the time of maximum product formation (30-40 rnin), a slower pseudo-first-order decomposition was observed (tip 1h) than in their absence. The radical scavenging properties (19, 20) of the organic modifiers and the sigmoidal kinetic plots obtained in their absence together with the observed influence of added oxygen suggest that radical species may be involved in both the product formation and degradation reactions. These results also suggest that inclusion of organic modifiers in the mobile phase is not compatible with optimal performance of the detector. Influence of Added Salts. The rate of formation of the major product in the CDDP/HS03- reaction decreased with increasing concentrations of NaC1, probably due, a t least in part, to a common ion effect (21). The addition of sodium acetate to the reaction mixture not only increased the rate of product formation but also significantly decreased the rate of decomposition of the major product (Figure 3). This rate enhancing effect may arise through an electrophilic catalytic contribution as observed in similar systems (22-24). Increases in product stability were also afforded by the addition of both
-
0
4
8
I2
16
20
24
28
lime (hours1
Flgure 3. Stabilizing effect of NaOAc (10 mM (A), 5 mM (B), 1.0 mM (C), 0.5 mM (D), 0.1 mM (E)) on the major reaction product formed between NaHS0, CDDP. Sample F contains 10 mM NaCi [condltions as in Figure 21.
+
citrate or phosphate salts to the diluent. Whereas citrate exerted a rate increasing effect on product formation, phosphate did not enhance the product formation rate. In all cases, the pH of the reaction mixture (4.5-5.5) was controlled primarily by the buffer capacity of the reagent (HSO3-/S03-) itself. NaC104 and NaN03 had the least effect on product formation kinetics. Since these salts exhibited major incompatibilities with the chromatographic system, they were not investigated further. Role of Sulfite Species. As the fraction of HSOf in the stock solution (0.1 M (bi)sulfite) diminished,both the product yield and the rate of formation decreased. Samples in which sodium sulfite (pH 7.5) was used as reagent required >400 min to reach a maximum product yield which was -10-fold less than that observed with bisulfite (pH 4.5). Influence of Added Metal Ions. When compared to samples run simultaneouslywithout added metal ions, ZnC12, MnC12,and CuS04 did not significantly alter the kinetic behavior of the cisplatin/bisuKte reaction. However, a dramatic increase in the product formation rate was observed when traces of the oxidizing agents KMn04 or KzCr2O7were added to the reaction mixture (Figure 4). Maximum yield was obtained in 2-3 min with added K2Cr207and 5-7 min with added KMn04 as contrasted to 30-40 min in the absence of these additives. For equimolar concentrations of reagents, yield in the presence of added K2Cr20, was -20% greater than in the presence of KMn04. Based on the significant increase in formation rate and product yield, solutions containing K2Cr207were used as the diluent in all subsequent studies. The product distribution in the cisplatin/bisulfite reaction mixture was not affected by the addition of dichromate (Figure 1). In the absence of added oxidizing agent, kinetics and product yield were highly irreproducible due to metal ion contamination of laboratory water supplies. Reagent Considerations. The effect of variable NaHS03 concentrations on the product yield (determined 1min after combination of reagents; Figure 5) indicated that maximum
494
ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984 Table I : Selected platinum complexes used in these
I
investigations.
I
c~ - d ic hlorod ia m mi ne pla t i nurn (II)
H2 N,
CDDP;cisplatin; NSC 119875
HSJ.
/cI
ch-dichloro(ethy1enediamine)platinum (11) Pt (en)C12
(NNPt\CI H2
c&-dichlora(l,2-diaminocyclohexane)platinum (11) DACC12; NSC 194814 __
trans-dichloradiammineplatinum(II ) NH3
t D D P ; NSC 131558 1
I CI,
/CI
-
H2
(1,2-diaminocyclahexane)malonatoplatinum(l1) DACMAL; NSC 224964
potassium tetrachloroplatinats(ll)
K2PtC14; P t C I i
,2K+
H2
0-c,
0
0 (malonato)diammincplatinurn(II)
,O-C: rlPt, H3N
,CH2 0-Cy
M A L \ NSC I 4 6 0 6 7
0 ~~
I
H2
s-dichloro-trans-dihydroxobis(isopropyiamine)platinum(lV)
H$\
CHIP; NSC 2 5 6 9 2 7
H3N
~
~~
(hydroxymalonato)diammineplatinum(ll) ,CHOH
0-C,
0
MALOH, NSC 201343
I, I -cyclobutanedicarboxylatodiornrnineplatinum(1 I)
Pt(i-Pr)2C12; NSC 215154
CBDCA; NSC 241240
\ I
60
40
10-c: ,Pt\
G-dichloro-bis(isopropy1amine)platinurn(ll)
\ 20
~~~
0
H 2 o.H (CKJ12CH-N, 1 /CI Pt (CH3)2CH-N’ ‘CI OH
0
~~~~
BO
I
100
1 20
time ( m i n )
Flgure 4. Effect of K,Cr,O, (1.0 X lo-’ M (0)and 1.0 X M (H)) added to the standard NaHSO, CDDP reaction (A)on the formation rate of the major product (conditions as in Figure 2).
+-
yield began to approach a limiting value at 1.6 mM NaHS0,. Concentrations greater than 2.5 mM could not be examined due to the adverse effects of bisflite on the solvent generated anion exchange column. As the concentration of KzCrz07was varied, yields were greatest at the lower concentrations ((0.5-2.5) X lO* M) and began to decline at concentrations X . 5 X lo* M. Through the selective addition of KzCrZO7to either the stock NaHSO, solution or the bulk diluent reaction mixture, an initial interaction between K2CrZO7and CDDP (15-20 s), prior to NaHSO, addition, was shown to be a requirement for
the rapid formation of the final product. Samples in which the K2Cr207was added only to the NaHS03 reagent stock solution formed product no faster than those run in the absence of KZCrzO7. The platinum(1V) compounds CHIP and KzPtCls (Table I) were inert under the reaction conditions employed (over a period of several hours) suggesting it to be unlikely that the role of dichromate is to oxidize CDDP to a platinum(1V) species prior to reacting with bisulfite. However, kinetic data are consistent with a mechanism in which KzCr2O7reacts with CDDP to form an activated species which then combines rapidly with bisulfite to give the UV absorbing complex(es). In light of recent postulations of the intermediacy of platinum(II1) species (25-27) in the oxidation of platinum(I1) compounds, it is tempting to suggest the existence of such species in this system. Post-Column Reactor. Design of Reactor. The bisulfite post-column derivatization reaction for cisplatin was initially examined with a traditional air-segmented flow reactor (28). This reactor design was abandoned when the surfactant properties of the mobile phase prevented the complete removal of the air bubbles from the flowing stream prior to detection. The reactor was redesigned (10) utilizing separate HPLC pumps to obtain a more constant, uniform addition of the reagents to the flowing stream. The use of a packed bed reactor (29-32) generated excessive back pressure with significant band broadening and did not provide sufficient reaction delay time for maximum yield. Recently, Englehardt et al. (18)have shown that extensive lengths of Teflon tubing may be used to achieve significant reaction delay time in a post-column reactor if the tubing is first knit on a spool to give a cylindrically braided coil. No significant band broadening was observed in chromatograms obtained before and after the addition of 47.8 m of braided (0.3 mm i.d.) Teflon tubing to the reactor. A band broadening effect was observed (Figure 6), however, when large internal
ANALYTICAL CHEMISTRY, VOL. 56,NO. 3,MARCH 1984 495 42
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CDDP
C '-*/
T
CDDP
z
I
444 0
5
10
0
I
I
I
I
5 1I I
IO I
time ( m i n u t e s )
Figure 6. Effect of large (0.79mm) (a)and small (0.3mm) (b) internal diameter Teflon tubing used in the bralded segments on band broadenlng in the post-column reactor at equivalent reaction delay times M K2Cr20,); delay (reactor as in ref 10 (3.3mM NaHSO,, 2.6 X braids (a) At, = 3.2 m X 0.3 mm i.d., At2 = 7.9 m X 0.79 mm i.d.; (b) Af, = 3.2 m X 0.3 mm i.d., Af2 = 44.6 m X 0.3 mm i.d.; chromatographic conditions as In Figure 1 (mobile hase = 10 mM citrate (pH 4.5,lo-' M HTAB), 290 nm, 2.52 X 10- M CDDP). 2
6
I
I
I
I
I
10
14
18
22
28
Flgure 5. Effect of variable concentrations of NaHS0, and K,Cr,O,
on the yield in the reaction of NaHSO, with CDDP. (chromatographic M CDDP; [K2Cr20,] = 0.0 M conditions as in Figure 2; 2.52 X (A), 0.5 X 10" M (B), 1.0 X 10" M (C), 2.5 X 10" M (D), 10.0 X 10" M (E), 25.2 X lo-' M (F), 50.0 X IO-' M (G),101.0 X lo-' M (H)). diameter tubing (0.79mm) was substituted for small internal diameter tubing (0.33mm) without altering the reaction time delay. If the length of braided tubing in the flowing stream was varied after reagent addition (At,), the response of the reaction detector to constant injections of CDDP reached a plateau between 2 and 5 min with maximum signal/noise ratios at times greater than 4.5 min. The effect of altering the time between the addition of the two reagents (Atl) did not produce as significant an effect on the yield as observed for changes in At,. Delays of -26 s and 4.7 min were established as optimal values for mixing times after introduction of dichromate and bisulfite reagents, respectively, for all later studies. pH and Reagent Effects. Alterations in the pH of the mobile phase would be expected to affect the reaction yield since response was shown (see previous kinetic section) to be a function of the bisulfite concentration. Maximum response for the post-column reactor (and maximum signal to noise ratio) was obtained with pH 4.5 mobile phase and 3.3-5.0 mM NaHSO,. Utilizing this optimum concentration (3.3 mM) of NaHSO, with pH 4.5 mobile phase, a constant response to variable concentrations of K,Crz07 was observed between 1.0 X and 2.5 X M while signal to noise was maximum between 2.0 X and 2.6 X M dichromate. Response to Platinum Compounds. Employing the optimum conditions defined for CDDP (see Figure 6 ) , the response of the post-column reaction detector to various platinum-containing compounds, separated on an analytical column, was determined. Response to derivatized CDDP was linear 0, = 0.491~+ 0.143,correlation = 0.999)with excellent reproducibility and precision (&2.5%) over the entire con-
B
centration range examined (2.55X to 4.0 X 10" M; 0.077 to 12.0 wg of CDDP/mL) with a detection limit of 40 ng of CDDP/mL (SIN = 3). Equimolar (2.52X 10" M)solutions of the platinum compounds listed in Table I were prepared to test and compare the reactor response to other platinum complexes. The cisdichloro compounds (DACCl,, CDDP, Pt(en)Cl,) were chromatographed simultaneously with the previously described solvent-generated anion exchange system utilizing a pH 4.5 or 5.0 citrate buffered mobile phase. A comparison of direct UV detection at 300 nm (no derivatization) and reactor response at 290 nm (Amm) (Figure 7) demonstrated a 60-to 80-fold increase in sensitivity after derivatization. Pt(i-Pr),Cl, chromatographed with a k'= 24-29 on the solvent-generated anion exchange system and was not investigated in any greater detail. trans-DDP also reacted in the bisulfite system; however, the increase in sensitivity over direct UV detection could not be accurately measured as trans-DDP eluted with the solvent front. When chromatograms of the underivatized substituted platinum malonates (MAL, MALOH, CBDCA, DACMAL) were compared to those of the corresponding compounds after derivatization (not shown), it was found that derivatization resulted in detectability at 290 nm whereas no absorbancewas observed for the underivatized samples at this wavelength. However, when compared to direct UV detection at 240 nm, post-column derivatization offers only a 2-to 4-fold increase in sensitivity. The major function for derivatization of the malonato-containing platinum complexes is to shift the detection wavelength from 240 nm to 290 nm. The reaction conditions employed (optimized for cis-dichloro compounds) may not be optimal for the (substituted)malonato complexes since these compounds are reported to undergo substitution reactions at a rate slower than the cis-dichloro complexes (33). No attempts were made, however, to optimize conditions for the (substituted)malonats. Employing the reactor conditions utilized in the analysis of CDDP, a response was observed to
498
ANALYTICAL CHEMISTRY, VOL. 56, NO. 3,MARCH 1984
I
I
-5 0 0
m
0)
0 C 0
n L
v) 0
n 0
O
W time
(I
0
5
io
1
I
I
nutes)
\h
IC
-- ---
0
Flgure 7. Comparison of UV detection (300nm) after HPLC separation (a) and reactor response (290 nm) to derivatized platinum (b) for a mixture of the cis dichloroplatinum complexes (conditions as in Flgure 6 with 2.52 X IO-' M DACCI, (l),2.52 X lo-' M Pt(en)CI, (2), 3.02 x 10-5 M CDDP (3)).
Table 11. HPLC Detection Limits for Selected Platinum(I1) Compounds
platinum compounde CDDP Pt(en)CI, DACCI, MAL MALOH CBDCA DACMAL
response to underivatized platinate response to wave- detection derivatized length, limit, platinate,f nm pgg/mL ng/mL 300 300 300 24 0 240 240 240
20b 207: 5" 5a 20" 20"
40 40-50' 40-50' 260d 300d 1200d 1200d
a Determined by using pH 4.5 acetate (10 mM) on 1 5 cm 5 pm Hypersil ODS column. Determined by using pH 4.5 citrate (10 mM, M HTAB) on 10 cm, 5 pm Hypersil ODs column. Estimated based on reactor M injection (20 pL). Estiresponse to 2.52 x mated based on reactor response to 2.52 X 10'' M injection (20 pL). e See Table I for structures of the respective platinum compound. Derivatized in post-column reactor using the dichromate/bisulfite reaction with detection at 290 nm.
5
10
time (minutes)
Figure 8. Post-column reactor response to plasma ultrafiltrate blank (a) and plasma ultrafiltrate containing CDDP (b). CDDP peak is the reactor response to 5.17 ng of platinum (conditions as in Figure 6 with mobile phase = 10 mM citrate (pH 5.0, lo-' M HTAB)).
greatest sensitivity for analysis of the cis-dichloro complexes (DACC12, Pt(en)C12,CDDP) followed by MAL, MALOH > CBDCA, DACMAL. This comparison assumes the diminished response to the (substituted)malonato complexes was due to diminished yield under the reaction conditions optimized for CDDP and was not due to other factors which would result in a lowered absorptivity of the derivatized platinate. The utility of the reaction detector in monitoring the kinetics of cisplatin degradation in plasma was also evaluated. From an initial concentration of 4.0 kg/mL, levels of CDDP in plasma were followed (for -350 min) to a level of 150 ng/mL (Figure 8). The pseudo-first-order half-life of CDDP in this medium was 1.4 h at 37 "C, in good agreement with previous reports (16). Thus, an on-line reaction detector is available which is suitable for sensitive detection of cisplatin and other divalent platinates (differing in ligand composition) in clinical specimens. Registry No. CDDP, 15663-27-1;tDDP, 14913-33-8;K2PtCI,, 10025-99-7;K2PtC&,16921-30-5;CHIP, 62928-11-4;Pt(i-Pr)&l2, 41637-05-2;Pt(en)C12,14096-51-6;DACC12,52691-24-4;DACMAL, 52351-07-2; MAL, 38780-43-7; MALOH, 52260-82-9;CBDCA, 41575-94-4; sodium bisulfite, 7631-90-5.
-
LITERATURE CITED the divalent platinate, PtC1,2-, but not for the tetravalent platinum compounds PtCle2- and CHIP. In Table 11, the detection limits for both the direct UV quantitation (after HPLC separation) and the dichromate/ bisulfite post-column reaction detector response to selected platinum(I1) complexes are compared. As none of the (substituted)malonato complexes exhibited a well-defined , , ,X the detection limits for the underivatized analytes were determined a t 240 nm where the base line noise levels were dimiihed. Following the trend predicted by the relative rates of substitution (Cl- > -OR (21)),the reactor demonstrated
(1) Prestakyo, A. W., Crooke, S. T., Carter, S . K., Eds. "Cisplatin: Current Status and New Developments"; Academic Press: New York, 1980. (2) Patton, T. F.; Himmelstein, K. J.; Belt, R.; Bannister, S . J.; Sternson, L. A.; Repta, A. J. Cancer Treat. R e p . 1978, 62. 1359-1362. (3) Belt, R. J.; Himmelstein, K.; Patton, T. F.; Bannister, S . J.; Sternson, L. A.; Repta, A. J. Cancer Treat. R e p . 1979, 63,1515-1521. (4) Himmelstein, K. J.; Patton, T. F.; Belt, R. J.; Taylor, S.;Repta, A. J.; Sternson, L. A. Clin. Pharmacol. Ther. 1981, 2 9 , 658-664. (5) Hoeschele, J. D..Warner Lambert Corp., Ann Arbor, MI, unpublished data. (6) Bannister, S. J., Ph.D. Thesis, The University of Kansas, 1982. (7) Riley, C. M.; Sternson, L. A,; Repta, A. J.; Siegler, R. W. J . Chromat o g . 1982, 229, 373-386. (8) Chang, Y.; Sternson, L. A,; Repta, A. J. Anal. Lett. 1978, B f f , 449-459.
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RECEIVED for review April 25, 1983. Accepted October 11, 1983. Abstracted in part from the Ph.D. thesis of K.C.M. This work was also supported in part by a University of Kansas Honors Fellowship (to K.C.M.) and Grants CH-149 from the American Cancer Society and CA-09242 and CA-24834 from the National Cancer Institute (National Institutes of Health).
Determination of Neutral Sugars in Plankton, Sediments, and Wood by Capillary Gas Chromatography of Equilibrated Isomeric Mixtures Gregory L. Cowie and John I. Hedges* School of Oceanography, University of Washington, WB-10, Seattle, Washington 98195
A reproducible technique is described for extraction and quantitative anaiysls of neutral monosaccharides from a variety of wild natural sample types, requiring as ifflie as 10 mg of total organic matter. Acid hydrolysis yields monomeric sugars which may exist in up to five isomeric forms when in solution. Lithium perchlorate is used to catalytically equiiibrate sugar isomer mixtures in pyridine prior to conversion to their trimethyisiiyi ether derivatives. Analysis is carried out by use of gas-liquid chromatography on fused-silica capillary columns. Quantlflcation on the basis of a single clearly resolved peak for each sugar is made possible by the equiilbration step. Sugar losses and optimal conditions for maximum reproducible sugar recovery are determined for each extraction stage.
Carbohydrates are major structural and storage compounds in both terrestrial and marine organisms, representing the largest fraction of the photosynthetically assimilated carbon in the biosphere. The purpose of this study was to develop a sensitive and reproducible technique for the hydrolytic extraction and quantitative analysis of complex monosaccharide mixtures in a broad range of solid, natural samples including plant tissues, plankton, and sediments. Further objectives were that the technique should employ commonly available instrumentation, involve a minimum of chemical manipulation prior to analysis, and be as straightforward and rapid as possible. Among the major problems associated with published quantitative carbohydrate methods have been the large variety and frequently poor reliability of techniques used for the extraction, identification, and measurement of carbohydrates 0003-2700/84/0356-0497$01.50/0
from natural samples (1). Due to the lack of a standard extraction technique and, in most cases, information on sugar recoveries, direct comparison of the results of different studies has proven difficult or impossible. Gas-liquid chromatography (GLC) has been a commonly used technique for monosaccharide analysis but requires formation of volatile derivatives since free sugars are insufficiently volatile for direct analysis. Both trimethylsilyl (Me,%) ethers (2-7) and trifluoroacetate (TFA) esters (49) have proven useful for analytical purposes. However, when in solution free sugars may exist in as many as five different forms (one acyclic form and two anomers for each of the fiveand six-membered ring forms). This leads to a complex multiplicity of peaks which has proven difficult to resolve. In addition, TFA derivatives have problems of poor sample stability and on-column losses (9). Reduction of monosaccharides to their corresponding alditols followed by the formation of MeaSi ether or acetate derivatives (10-14) avoids the problem of peak multiplicity by removing the carbonyl group which is normally involved in ring formation through internal glycosidic bonds. Peaks are therefore much simpler to resolve since only one peak is obtained for each sugar. This method, however, involves significant chemical manipulation of the sugars and may lead to information-loss because certain sugar pairs (e.g., lyxosearabinose, gulose-glucose) yield the same alcohol as their reduction product. Ketoses and aldoses present in the same sample can also yield the same alcohol (e.g., sorbitol from glucose or fructose). In addition, ketoses always yield more than one alcohol (e.g., fructose produces both sorbitol and mannitol). Conversion of the free sugars to their oximes followed by formation of Me& ether derivatives (2, 15, 16) also reduces the problem of peak multiplicity (two possible 0 1984 American Chemical Society
