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Department of Toxicology, Institute of Pharmacology and Toxicology, University of Saarland, D-66421 Homburg (Saar), Germany. Portions of this work were published in the proceedings of the 34th International TIAFT Meeting, August 11–15, 1996, Interlaken, Switzerland (Toennes SWH, Maurer HH. Immobilization of ß-glucuronidase and arylsulfatase: which are the advantages of column packed immobilizate for the cleavage of conjugates in analytical toxicology? In: Sachs H, Bernhard W, Jeger A, eds. Proceedings of the 34th International TIAFT Meeting in Interlaken. Leipzig, Germany: Molinapress, 1997:92–6) and the 35th International TIAFT Meeting, August 24–28, 1997, Padova, Italy (Toennes SWH, Maurer HH. Column packed immobilized ß-glucuronidase and arylsulfatase for the cleavage of conjugates—stability, reusability and application in toxicological routine analysis. In: Ferrara SD, ed. Proceedings of the 35th International TIAFT Meeting in Padova. Padova, Italy: Centre of Behavioural and Forensic Toxicology, 1997:227–40).
a Author for correspondence. Fax 49-6841-16-6051; e-mail Hans.Maurer{at}med-rz.uni-sb.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionDetermination of free morphine....References |
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Methods: ß-Glucuronidase (GRD; EC 3.2.1.31) and arylsulfatase (ARS; EC 3.1.6.1) were purified and coimmobilized on an agarose gel matrix and packed into columns.
Results: In columns packed with GRD and ARS, the test conjugates 4-nitrophenyl glucuronide and 4-nitrophenyl sulfate added into urine could be completely cleaved within 25 min. Even the relatively stable morphine conjugates could be completely hydrolyzed within 60 min in authentic urine samples. Therefore, an incubation time of 1 h is recommended. Enzyme inhibition by matrix or by rather high concentrations of acetaminophen conjugates was tested and found to be up to 50%. However, a large excess of GRD and ARS was used. The immobilizate columns could be reused for at least 70 incubations and had a storage stability of at least 12 weeks. Carryover of analytes in reused columns could be avoided by rinsing with 200 mL/L methanol in acetate buffer. Thus, five drugs known to be contaminants added in very high concentrations into urine could be completely removed from the columns. A study on the applicability in systematic toxicological analysis showed that 120 different drugs and/or their metabolites could be detected in 35 different authentic urine samples.
Conclusions: Use of immobilized and column-packed GRD and ARS is an efficient alternative for the cleavage of urinary conjugates in clinical toxicology.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionDetermination of free morphine....References |
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To improve the enzymatic hydrolysis procedure, we developed a fast, inexpensive, and easy-to-handle procedure based on immobilized GRD and ARS. In previous studies, several attempts had been made to immobilize GRD and ARS. The aim in most of these studies was to compare different immobilization techniques (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Application of immobilized GRD and/or ARS for online cleavage of conjugates in pre- or postcolumn reactors in HPLC has also been described (10)(16)(17)(18)(19)(20). Previous studies on the application of immobilized enzymes for cleavage of conjugates in analytical toxicology showed that this was no alternative to acid hydrolysis because an incubation time of 24 h was necessary (21)(22). Furthermore, in these studies only GRD was immobilized, so that sulfate conjugates, e.g., of steroids, could not be cleaved.
Several crude enzyme preparations are on the market. Preparations of Escherichia coli or bovine liver lack ARS and therefore can not be used for the cleavage of sulfate ester conjugates. Preparations of the digestive juice of the snail Helix pomatia, which contain both enzymes, are widely used. GRD and ARS from Patella vulgata were evaluated only as second best because certain conjugates were not cleaved (23) and the optimal pH values differed in a broader range (24)(25).
In this report, a procedure is presented for the fast cleavage of conjugates in urine using immobilized GRD as well as ARS packed in easy-to-handle columns.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionDetermination of free morphine....References |
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procedures
Determination of enzyme activities.
For determination of the
activity of GRD, 0.28 mL of the enzyme solution was incubated at
35 °C with 2.52 mL of a solution of 11.1 mmol/L NPG in 0.1 mol/L
acetate buffer, pH 5.2, containing 0.5 mol/L sodium chloride (standard
acetate buffer). Five 0.5-mL aliquots of the incubation mixture were
taken consecutively, and free 4-nitrophenol (NP) was determined after
addition of 1.0 mL of 0.5 mol/L NaOH at 405 nm using an Eppendorf
UV-Vis photometer (Eppendorf). Linear regression indicating the
increase of free NP in the assay was calculated. The activity was
calculated from the slope. The activity of ARS was determined in the
same way, using 38.0 mmol/L NPS as substrate. For the determination of
GRD and ARS activities in urine, the substrates were added to sterile
filtered blank urine (pH 5.2).
The enzyme activities of immobilized GRD and ARS were determined in a similar manner: 0.5 mL of an immobilizate suspension was mixed and incubated at 35 °C under gentle agitation with 4.5 mL of substrate solution (NPG or NPS in standard acetate buffer or urine). Five 0.9-mL aliquots of the incubation mixture were taken consecutively and centrifuged at 15 100g for 10 s. Aliquots (0.5 mL) of the supernatant were mixed with 1.0 mL of 0.5 mol/L NaOH, and the absorbance was measured at 405 nm. Again, linear regression indicating the increase of free NP in the assay was calculated. The activity was calculated from the slope. For our studies, we defined the activity of 1 U of GRD or ARS as that amount of enzyme that hydrolyzes 1 µmol NP conjugate/min (35 °C at pH 5.2).
The protein content of enzyme solutions was determined by the Bio-Rad protein assay based on the Bradford method (26) using a photometer SP8–500 from Pye Unicam. The specific enzyme activities could thus be calculated as enzyme activity per milligram of protein (U/mg).
Purification of the crude GRD/ARS enzyme preparation.
Desalting and buffer exchange of 2 mL of the crude BM GRD/ARS
preparation (containing approximately 40 U each of GRD and ARS) was
performed using a PD10 column from Pharmacia after equilibration with
25 mL of 20 mmol/L
bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane, pH 7.0
(BisTris buffer). The pH of the eluate was adjusted to 7.0 by dilution
with the BisTris buffer.
For anion-exchange chromatography, a Pharmacia FPLC system [two P-500 pumps, one P-1 peristaltic pump, a GP-250 gradient controller, and a UV-1 ultraviolet (UV) detector equipped with 280 nm filter] was used with four connected 1-mL Pharmacia HiTrap Q columns. All chromatographic steps were performed at a flow rate of 1 mL/min, and the UV absorbance at 280 nm of the effluent was recorded. The column was equilibrated once with 10 mL of BisTris buffer, once with 15 mL of BisTris buffer containing 2 mol/L NaCl, and thereafter with 20 mL of BisTris buffer. The diluted enzyme solution was injected using the peristaltic pump. After removal of unbound contaminants with BisTris buffer, GRD and ARS were eluted with BisTris buffer containing 150 mmol/L NaCl. Before reequilibration of the column, strongly bound contaminants were removed with BisTris buffer containing 2 mol/L NaCl and further contaminants with five injections of 0.5 mL of 2 mol/L NaOH.
The GRD- and ARS-containing eluate was concentrated using a Centrisart I ultrafiltration device with a 10-kDa polyethersulfone membrane from Sartorius. The buffer of the ultrafiltrate was exchanged with a PD10 column to 0.1 mol/L 3-morpholinopropanesulfonic acid, pH 7.5, containing 80 mmol/L CaCl2 (coupling buffer). This enzyme solution was stored at 4 °C.
Immobilization of GRD and ARS.
Affi-Gel 10 was washed with 10
column volumes of cold 1 mol/L HCl and 10 column volumes of cold
deionized water using a VacMaster 10 vacuum manifold from ICT. The wet
gel cake was then mixed in a ratio of 2:1 (1 mL of gel cake + 1 mL of
enzyme solution) with cold purified enzyme solution containing ~10
kU/L GRD and 10 kU/L ARS. For the immobilization process, the mixture
was rotated overnight at 4 °C in an end-over-end Reax 2 shaker from
Heidolph. The supernatant was recovered, and after concentration, it
could be reused for further immobilizations. The immobilizate was
consecutively washed once with coupling buffer, once with 1 mol/L NaCl,
pH 7.5, and thereafter with standard acetate buffer until no more
enzyme activity was detectable in the eluate. The final
immobilizate was stored in standard acetate buffer at 4 °C.
Packing of columns with immobilized GRD and ARS (immobilizate
column).
For preparation of immobilizate columns, empty Chromabond
3-mL glass solid-phase extraction (SPE) cartridges from Macherey &
Nagel were used. Before use, they were deactivated by incubation with
50 g/kg trimethylchlorosilane in toluene for 1 h and dried
for 1 h at 100 °C. Columns were equipped with a polyethylene
frit and filled with 3 mL of immobilizate in standard acetate buffer.
The gel bed was protected by a second frit. The immobilizate
columns were stored at 4 °C.
Handling of column-packed immobilized GRD and ARS for cleavage of
conjugates.
A specially designed apparatus (Fig. 1
) was used for cleavage of conjugates in urine. An immobilizate
column was connected to a liquid handling system consisting of a buffer
reservoir, a three-way valve, and a thermostated transfer line to the
column. The column was thermostated in an aluminum block at 35 °C.
Before incubation, urine samples were sterile filtered, and the pH was
adjusted to 5.2. One milliliter of urine was injected onto the column,
using a disposable syringe, for incubation. Afterward, the urine was
eluted from the column with 10 mL of standard acetate buffer by
switching the injection valve to the buffer reservoir.
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Determination of NPG, NPS, and NP in urine.
NPG, NPS, and NP
were determined simultaneously by isocratic HPLC using an Hewlett
Packard (HP) HP 1050 HPLC system (20-µL sample loop, UV detector, and
a DOS/Windows computer with the ChemStation software HP A.02.05.). The
three analytes were separated within 16 min from the urine matrix on a
Merck Purospher RP18 column (250 x 4 mm i.d.; 5 µm bead size).
The mobile phase consisted of 700 mL/L 20 mmol/L
(NH4)2HPO4
buffer (pH 6.0)-300 mL/L acetonitrile at a flow rate of 0.8 mL/min. The
detection was optimized using a wavelength program with 305 nm for NPG,
278 nm for NPS, and 316 nm for NP. The samples were diluted 3:1 (2 mL
of sample + 1 mL of acetonitrile) with acetonitrile, and 100 µL of
these mixtures was injected into a 20-µL sample loop. For correction
of matrix interferences, the UV chromatogram of the corresponding blank
urine was subtracted from that of the sample. Quantification was
carried out using a five-point calibration: 0.003, 0.071, 0.143, 0.714,
and 1.429 mmol/L for NPG and NPS; and 0.006, 0.143, 0.286, 1.429, and
2.857 mmol/L for NP added to a blank urine sample. The precision and
accuracy of the method were determined by measuring three control
concentrations (0.174, 0.357, and 1.071 mmol/L for NPG and NPS; and
0.029, 0.714, and 2.143 mmol/L for NP added to blank urine) five times.
Determination of free morphine in urine.
Four milliliters of
0.1 mol/L K2HPO4 and 0.1 mL
of a methanolic 1 mg/L
morphine-d3 solution were added to 2
mL of urine sample. This mixture was extracted at a flow rate of 6
mL/min, using a BenchMate sample preparation robot from Zymark and a
3-mL Chromabond C18ec 200 mg SPE column from Macherey & Nagel. The
column was conditioned with 2 mL of methanol and then with 1 mL of
deionized water at a flow rate of 30 mL/min. After addition of the
urine sample, the column was rinsed with 1 mL of deionized water and 1
mL of a 50 g/L NaHCO3 solution, followed
by 1 mL of deionized water at a flow rate of 30 mL/min. The column was
then dried with nitrogen (300 kPa) for 5 min. The analyte was eluted
with 3 mL of dichloromethane-acetone (1 mL of dichloromethane + 3 mL of
acetone) at 6 mL/min. The extract was evaporated to dryness and
derivatized with 100 µL of pyridine-acetic acid anhydride (3 mL of
pyridine + 2 mL of acetic acid anhydride) for 30 min at 65 °C. The
derivatization mixture was evaporated, and the residue was dissolved in
100 µL of methanol. One microliter of the redissolved residue was
injected into an HP 5890 Series II gas chromatograph equipped with an
HP autosampler 6890 and coupled to a HP 5989 Engine B mass
spectrometer. The gas chromatography (GC) conditions were as follows:
splitless injection mode; HP-1 capillary column (12 m x 0.2 mm
i.d.); 280 °C injection port temperature; helium as carrier gas at a
flow rate of 1.0 mL/min; programmed column temperature, 100–310 °C,
ramped at 30 °C/min; 3 min initial time; and 4 min final time. The
mass spectrometry (MS) conditions were as follows: 70 eV ionization
energy; 220 °C ion source temperature; capillary direct interface
heated at 280 °C; selected-ion monitoring mode with the selected
ions m/z 327 and 369 for acetylated morphine and
m/z 330 and 372 for the acetylated internal standard
morphine-d3. Quantification was based
on an eight-point calibration curve with 83, 104, 208, 1038, 3114,
5190, 10 380, and 15 570 µg/L of morphine in blank urine. The
accuracy and the intraday precision of the quantification method were
determined by measuring three control concentrations (519, 2076, and
8304 µg/L morphine) in blank urine five times.
Influence of urine matrix or xenobiotics on the activities of GRD
and ARS.
Blank urine samples from 10 different persons (5 females
and 5 males) were collected in the morning. Samples of all these blank
urines were supplemented with either 11.1 mmol/L NPG or 38.0 mmol/L
NPS. Aliquots of GRD/ARS immobilizate were dispersed in each of these
urinary substrate solutions and incubated as described for enzyme
activity determination. These enzyme activities were compared with
those obtained in standard acetate buffer containing the same
concentration of NPG and NPS and the same amount of enzymes. The
differences between the enzyme activities in buffer and urine indicated
the inhibiting effect of endogenous compounds in urine.
For testing the influence of high concentrations of xenobiotic conjugates on the GRD/ARS activity, urine samples after the ingestion of acetaminophen were used. After four healthy volunteers (one female and three males) were informed according to the declaration of Helsinki and gave written consent, they each received a single oral dose of 1 g of acetaminophen in the evening and the first urine samples were collected the next morning. As described above, GRD and ARS activities were determined in these four urine samples and were compared with the enzyme activities in standard acetate buffer.
Efficiency of conjugate cleavage with immobilizate columns.
The efficiency of the cleavage of NP conjugates in urine was tested
with blank urine supplemented with 10 mmol/L each of NPG and NPS
(NPG/NPS urine). Aliquots (1 mL) of this urine were incubated for
increasing time periods in an immobilizate column. The residual NPG and
NPS concentrations were then determined, and the hydrolysis yields of
both conjugates were calculated. For each incubation time, five
incubations were made. The efficiency of the conjugate cleavage was
also investigated with morphine conjugates. An authentic urine sample
of a morphine-treated patient was used containing 1490 µg/L free
morphine and 10 740 µg/L total morphine, indicating a conjugation
rate of 86%. The total morphine concentration was determined as
described for free morphine after acid hydrolysis according to the
method of Maurer and co-workers (27)(28).
Aliquots (1 mL) of the authentic urine (morphine-conjugate urine) were
hydrolyzed in an immobilizate column for increasing incubation times.
Five samples were incubated for each incubation time. The hydrolysis
yields of the morphine conjugates were calculated as the percentage of
free morphine in the total morphine concentration.
Influence of the substrate concentration on the hydrolysis
rate.
The NPG/NPS urine and dilutions of this urine with blank
urine were prepared to contain NPG and NPS in concentrations of 10, 5,
2.5, and 1 mmol/L. Five 1-mL aliquots of each of the four urine samples
were incubated in an immobilizate column for 5 min. The residual NPG
and NPS concentrations were determined as described above, and the
hydrolysis yields of both conjugates were calculated.
Reproducibility of conjugate cleavage with immobilizate
columns.
The reproducibility of the cleavage of conjugates with
immobilizate columns was tested with four immobilizate columns from
different batches. Aliquots (1 mL) of NPG/NPS urine were incubated in
the columns for 5 min (n = 5). In another series, 1-mL aliquots of
the morphine-conjugate urine were incubated in the columns for 15 min
(n = 5). The hydrolysis yields were determined in the eluates.
Stability and reusability of immobilizate columns in routine
analysis.
The stability of the immobilized enzymes and the
reusability of an immobilizate column were investigated with an
immobilizate column that was used continuously for a total of 70 h
under routine analysis conditions. This meant consecutive 1-h
incubations of morphine-conjugate urine samples up to the 70th h. In
regular intervals, the activities of the immobilized GRD and ARS were
determined by measuring the hydrolysis yields of NPG and NPS after
incubation of 1-mL aliquots of the NPG/NPS urine for 5 min in the
columns. The hydrolysis yields of the morphine conjugates were
determined in regular intervals after the 1-h incubations, and they
were used as an indicator of the reusability.
Carryover of analytes.
Blank urine samples were supplemented
with the known contaminants acetaminophen, dihydrocodeine,
phenobarbital (250 000 µg/L each), ibuprofen (25 000 µg/L), and
perazine (125 000 µg/L). Aliquots (1 mL) of this urine were
incubated in an immobilizate column and eluted with 10 mL of standard
acetate buffer. The column was then washed with 10, 20, or 30 mL of 200
mL/L methanol in standard acetate buffer and then twice with 10 mL of
standard acetate buffer. In the last eluate, the presence of the test
substances was checked by GC-MS as described below.
Eluate (10 mL; pH 5.2) was extracted with 10 mL of a mixture of dichloromethane-isopropanol-ethyl acetate (10 mL of dichloromethane + 10 mL of isopropanol + 30 mL of ethyl acetate). The remaining aqueous phase was then adjusted to pH 8.5–9 with 1 mol/L NaOH solution and extracted again. The combined organic extracts were evaporated, and the residue was dissolved in 100 µL of methanol and derivatized with 100 µL of an ethereal diazomethane solution for 10 min at room temperature (2). After evaporation, the residue was additionally derivatized with 100 µL of acetic acid anhydride-pyridine (3 mL of acetic acid anhydride + 2 mL of pyridine) for 30 min at 60 °C. After evaporation, the extract was dissolved in 100 µL of methanol containing 0.5 g/L methaqualone as internal standard. A 1-µL aliquot of this solution was injected into the GC-MS apparatus as described above. The GC-MS peak areas were measured with the HP G1034C MS ChemStation software by autointegration of the peaks in the mass fragmentograms of the following selected ions: m/z 235 for methaqualone, m/z 161 for methylated ibuprofen, m/z 232 for methylated phenobarbital, m/z 109 for acetylated acetaminophen, m/z 343 for acetylated dihydrocodeine, and m/z 339 for perazine. A methanolic solution containing 250 000 µg/L of each drug was used as the external calibrator.
Studies on the applicability in systematic toxicological analysis
(STA).
Thirty-five different authentic urine samples were selected
from urine samples submitted to our department for STA. STA was
performed either for emergency toxicology or for monitoring of drug or
medication abuse. Aliquots (1 mL) of these urine samples were analyzed
using three different sample preparation procedures: enzymatic
hydrolysis with an immobilizate column, enzymatic hydrolysis with
soluble BM enzyme solutions [H. pomatia enzymes (1000
Fishman units (FU)/mL of urine at 37 °C for 24 h)] or without
cleavage of conjugates. The samples were extracted at pH 8–9,
acetylated as described above, and analyzed by GC-MS in scan mode. The
data were evaluated as described in Refs.
(1)(29)(30)(31)(32).
statistics
Differences were tested for significance by either the Student
t-test or ANOVA, whichever applied.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionDetermination of free morphine....References |
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purification of the crude grd/ars enzyme preparation
Immobilization of commercial crude GRD/ARS preparations from
H. pomatia yielded poor enzyme activity per immobilizate
volume because other enzymes/proteins blocked binding sites of the
immobilization support. Therefore, the crude enzyme preparation was
first purified. Common gel filtration and hydrophobic interaction
chromatography did not yield the desired purification effect. However,
anion-exchange chromatography at pH 7 was suitable for purification.
The eluate fraction of such anion-exchange chromatography contained
84% ± 9% of the GRD and 91% ± 3% of the ARS (expressed as the
percentage of the enzyme activity in the crude GRD/ARS preparation).
Only 34% ± 2% of the total protein amount of the crude preparation
was retained, so that the relative amount of GRD/ARS markedly
increased. After buffer exchange and a concentration step, the enzyme
activity in the final coupling buffer used for immobilization was 72%
± 6% of that in the crude preparation for GRD and 75% ± 5% for ARS
with CVs <8% (n = 10 over 7 days). When stored in the
coupling buffer at 4 °C, both enzymes were stable for at least 5
months with an activity loss of only 1% ± 13% for GRD and 5%
± 4% for ARS (n = 5).
immobilization of grd and ars
Agarose beads activated with N-hydroxysuccinimide
(Affi-Gel) were chosen as a suitable immobilization support for the
purified enzymes. The coupling reaction was simple and yielded stable
amide bonds (33). The portion of the enzymes that was not
immobilized could be reused after ultrafiltration of the supernatant.
Thus, with minimized use of purified enzyme, the immobilizate
activities could be maximized. To determine the best coupling
conditions, GRD/ARS solutions with increasing enzyme concentrations
were used in several coupling reactions and the enzyme activities in
the resulting immobilizates, were measured. We found that coupling of
purified enzyme solutions containing 7–10 kU/L GRD and ARS yielded the
best enzyme activities (up to 3.3 kU/L GRD and 2.9 kU/L ARS) in the
immobilizate. The relation of the coupled GRD to ARS remained the same
as in the solution before coupling. The protein content of such
purified enzyme solutions was 20–30 g/L. GRD and ARS were stable
during the coupling reaction because the total of the enzyme activities
could be recovered in the immobilizate plus the supernatant. The
immobilizates were stable for at least 12 weeks without loss of enzyme
activity when stored at 4 °C in a solution of 2 mol/L ammonium
sulfate, pH 6.0, or in standard acetate buffer.
For investigation of the reproducibility of the coupling process, including purification, 2.0 mL of each of five independently purified enzyme solutions were incubated separately with ~4 mL of Affi-Gel. This volume was necessary for filling of the immobilizate columns. The coupling yields were 38% ± 4% for GRD and 33% ± 4% for ARS. The resulting immobilizate activities were 3.0 ± 0.2 kU/L for GRD and 2.4 ± 0.1 kU/L for ARS. The reproducibility of the immobilization including the purification step was satisfactory with a CV <11%.
handling of column-packed immobilized grd and ars for cleavage of
conjugates
The application of immobilizates in the form of suspended
particles in urine was not very efficient
(21)(22). Our aim was to use as much immobilized
enzyme activity as possible for a certain volume of urine. The simplest
way to do this was to put the immobilizate into empty 3-mL SPE glass
cartridges, which could be handled easily by most of the robots used
for automated SPE. The cartridges were silanized to minimize adsorption
of analytes and could be filled with up to 3 mL of immobilizate,
leaving 1 mL of volume for the urine sample to be cleaved. As shown in
Fig. 1
, a special aluminum heating block was constructed to ensure
thorough thermostatic control of the immobilizate in the column, the
urine samples, and the standard acetate buffer before they entered the
column. Thermostatic control was essential for good reproducibility of
the hydrolysis yields. The apparatus allowed the application of urine
with disposable syringes via one inlet of a three-way valve. The urine
could be eluted with standard acetate buffer, using a peristaltic pump
on the other inlet of the valve. A homemade flow adapter allowed simple
connection of the column to or disconnection of the column from the
liquid handling system. To minimize carryover of analytes in the flow
path, only polytetrafluoroethylene and nickel tubing were used.
determination of the test conjugates and their cleavage products in
urine
Determination of NPG, NPS, and NP.
The HPLC method for the
quantification of the NP conjugates NPG and NPS and their cleavage
product NP was simple and sufficient. The validation data of the
quantitative method are given in Table 1
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Determination of free morphine. The method for the
quantification of free morphine in urine was also simple and
sufficient. The validation data of the quantification method are given
in Table 2
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionDetermination of free morphine....References |
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efficiency of the conjugate cleavage with column-packed immobilized
grd and ars
For determination of the cleavage efficiency of column-packed
immobilizate, urine samples supplemented with 10 mmol/L each of NPG and
NPS were used (NPG/NPS urine). The conjugate concentrations were in a
molar concentration range as expected for acetaminophen conjugates
(36).
In addition, the efficiency of the cleavage of morphine conjugates in an authentic urine sample was tested because morphine is extensively conjugated (36) and its conjugates need strong conditions for enzymatic hydrolysis compared with other conjugates (3)(25)(37). Authentic urine samples (morphine-conjugate urine) were preferred in contrast to supplemented samples to have authentic conditions. Therefore, the cleavage yield in this urine was calculated from the morphine concentration after conjugate cleavage in relation to the total morphine concentration.
The cleavage efficiency was estimated by determining the time for
complete conjugate cleavage. Therefore, both types of urine samples
(n = 5) were incubated in an immobilizate column for increasing
times, and the hydrolysis yields were determined. The relative
hydrolysis yields of NPG, NPS, and morphine conjugates in urine in
relation to the incubation times are shown in Fig. 2
. After 15 min of incubation, >90% of both NP conjugates were
hydrolyzed. After 25 min, the NP conjugates were completely cleaved. As
expected, hydrolysis of the morphine conjugates took longer than
hydrolysis of the NP conjugates. However, after 60 min, the morphine
conjugates were almost completely hydrolyzed. After 15 min, only 40%
were cleaved, and after 30 min, 65% were cleaved. However, in
emergency cases in clinical toxicology, the latter cleavage yields
should be sufficient to find all relevant drugs, poisons, and/or their
metabolites. Nevertheless, an incubation time of 1 h is
recommended. This incubation time provides a drastic time reduction
compared with the 24-h incubation time recommended for immobilized GRD
by Buszewicz (22). The fastest method for cleavage of
morphine conjugates using soluble enzymes from P. vulgata at
high temperature needed 3 h for complete cleavage of conjugates
(25). Acceleration of enzymatic hydrolysis using a two- or
threefold amount of enzyme solution introduced much more matrix into
the extracts (3). Our immobilizate columns allowed 5- to
15-fold higher enzyme activities per milliliter of urine, producing
fast cleavage, clean extracts, and reduced cost by repetitive use.
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In a further study, the influence of the substrate concentration on the hydrolysis efficiency was investigated. Blank urine samples containing 10, 5, 2.5, and 1 mmol/L each of NPG and NPS were incubated for 5 min. The hydrolysis yields increased with decreasing conjugate concentrations: for NPG, 70% for 10 mmol/L, 80% for 5 mmol/L, 90% for 2.5 mmol/L, and 100% for 1 mmol/L; for NPS, 60% for 10 mmol/L, 70% for 5 mmol/L, 80% for 2.5 mmol/L, and 90% for 1 mmol/L. These results show that the hydrolysis yield of 1 mmol/L NPG and NPS after a 5-min incubation was the same as for 10 mmol/L after 15 min. The fact that the relative hydrolysis yield was higher at lower concentrations may be advantageous when low analyte concentrations must be analyzed.
reproducibility of conjugate cleavage with immobilizate columns
Urine samples were incubated for only short time periods (5 min
for NPG/NPS urine, 15 min for morphine-conjugate urine) because
variations attributable to the procedure should be detectable only if
complete cleavage is not achieved. For each of the four columns and for
each conjugate, five replicates were performed. Within the columns, the
CVs were 0.5–1.7% for cleavage of NPG, 0.4–3.0% for cleavage of
NPS, and 1.7–8.0% for cleavage of morphine conjugates. The overall
hydrolysis yields for the four different columns were 66–84% for NPG
with a CV of 7.9%, 72–86% for NPS with a CV of 6.2%, and 33–48%
for morphine conjugates with a CV of 9.4%. ANOVA showed significant
differences (P <0.001) between the columns. However, these
differences are of less relevance because a routine incubation time of
1 h is recommended and only the total cleavage yield is important
for toxicological analysis.
stability and reusability of immobilizate columns in routine
analysis
For the determination of the stability and the reusability of
column-packed immobilized GRD and ARS during routine operation, an
immobilizate column was used continuously for the cleavage of
conjugates up to a total of 70 h. During this time, a decrease of
the enzyme activities could be observed down to ~80% of the initial
values for GRD and ~65% for ARS. However, the hydrolysis yields of
morphine conjugates did not markedly change, indicating that the
columns had sufficient hydrolyzing capacity remaining. The immobilizate
column could thus be reused for at least 70 incubations, giving a
reproducible hydrolysis yield of morphine conjugates of 97% ± 5%
(n = 30). In the meantime, we have seen that the immobilizate
column could be reused for a much longer time when used only 8 h a
day and when stored at 4 °C overnight. The reusability of the
columns markedly reduces the costs, so that they are in the same range
as those for using soluble enzymes.
carryover of analytes in reused immobilizate columns
Carryover of analytes is a common problem that arises from the
reuse of apparatus or materials in analytical procedures. This means
that analytes from one sample can be adsorbed and afterward desorbed
into the next sample, producing false-positive results. To study how
analyte carryover could be avoided, blank urine samples supplemented
with high concentrations of ibuprofen, acetaminophen, dihydrocodeine,
phenobarbital, and perazine were incubated in an immobilizate column.
These substances were chosen because they are widely used and applied
in high doses. Furthermore, in our experience, they all are known for
analyte carryover. The work-up of the eluate was according to the
common STA procedure (1)(2).
Simple elution with 10 mL of standard acetate buffer was not sufficient
to completely remove these substances from the immobilizate column. To
study where the analytes were bound, columns packed with Affi-Gel but
without immobilized enzymes were used. Because no carryover was
observed in these columns, the analyte carryover should be attributable
to the immobilized proteins. Similar results had already been reported
for immobilized GRD from E. coli (16). However,
the authors could solve the problem by rinsing with mobile phase
containing 115 mL/L methanol. We had seen in preliminary studies that
the activities of GRD and ARS from H. pomatia were not
markedly influenced by methanol solutions up to 200 mL/L. Therefore, we
tried to eliminate analyte carryover in immobilizate columns by a
simple rinsing step between incubations. To study the efficiency of
this rinsing step, 10, 20, or 30 mL of 200 mL/L methanol in standard
acetate buffer was used for rinsing after incubation and routine
elution of the supplemented test urine. The analyte carryover was
expressed as the percentages of the analyte concentrations in the
standard acetate buffer eluate after the rinsing step compared with the
concentrations in the supplemented urine sample. As shown in Table 3
, one-time washing with 30 mL (10 column volumes) of a solution
of 200 mL/L methanol in standard acetate buffer was sufficient to
completely remove the test analytes from the column.
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studies on the applicability of the immobilizate columns for
cleavage of conjugates in sta
A study on the applicability of immobilizate columns for cleavage
of urinary conjugates in STA was performed to determine whether a broad
range of drugs and their conjugates could be detected in various
different authentic urine samples. Aliquots of these urine samples were
analyzed using three different sample preparation procedures: enzymatic
hydrolysis with an immobilizate column and enzymatic hydrolysis with
soluble enzymes or without cleavage of conjugates, followed by
extraction, acetylation, and full-scan GC-MS analysis. When
immobilizate columns were used, the following different drugs and/or
their metabolites (120 in all) could be detected in the 35 urine
samples: the sedative-hypnotics temazepam, nordiazepam, diazepam,
oxazepam, lormetazepam, lorazepam, bromazepam, clomethiazole,
diphenhydramine, and doxylamine; the opioids morphine, codeine,
dihydrocodeine, methadone, pethidine, and tramadol; the nonopioid
analgesics acetaminophen, acetylsalicylic acid, diclofenac, ibuprofen,
and metamizol; the antidepressants doxepin, amitriptyline,
trimipramine, opipramol, and viloxazine; the neuroleptics promethazine,
perazine, chlorprothixene, clozapine, and haloperidol; the
antiepileptics carbamazepine, phenytoin, primidone, and phenobarbital;
the drugs of abuse amphetamine, 3,4-methylene dioxyamphetamine,
3,4-methylene dioxyethylamphetamine, and cocaine; or the
antiarrhythmics verapamil and quinidine. The same compounds could also
be detected after conventional enzymatic hydrolysis using soluble
GRD/ARS. In several cases, the following substances were detectable
only after cleavage of conjugates: oxazepam, temazepam, lormetazepam,
morphine, codeine, hydroxypromethazine, norhydroxypromethazine,
nordoxepin, norhydroxydoxepin, hydroxytrimipramine, viloxazine, or
hydroxyviloxazine. Other substances, such as acetaminophen, ambroxol,
dihydrocodeine, codeine, hydroxyphenobarbital, or hydroxyphenytoin,
could also be detected in unhydrolyzed urine samples, but the peak
areas were much higher after cleavage of conjugates. This study
demonstrated that the cleavage of conjugates using immobilizate columns
was suitable for STA, so that in future studies, its applicability for
automated sample preparation can be tested.
comparison of the costs for conjugate cleavage using immobilizate
columns or soluble preparations
For comparison of the costs of our new procedure vs those of other
published procedures, the enzyme activities used in this report must be
converted to FU. One milliliter of the crude BM enzyme preparation
contained 100 000 FU of GRD as indicated by the manufacturer. This
corresponded to 20 U as defined in this report. This means that 1 U
corresponds to 5000 FU. The immobilizate columns each contained a total
enzyme activity of ~45 000 FU, which was used for the cleavage of
conjugates in 1 mL of urine. Meatherall (3) used 1000–5000
FU/mL of urine. One milliliter of the crude BM enzyme preparation costs
~$15 US ($0.15–0.75 US per milliliter of urine). Considering that
25% of the enzymes were lost during purification and that 3 mL of
Affi-Gel cost ~$15 US, one immobilizate column costs ~$ 20 US. One
column could be reused at least for 70 incubations, with a cost of
$0.30 US per hydrolysis of 1 mL of urine. Therefore, the costs are
comparable, although in our procedure the enzyme activity was up to
45-fold higher.
In conclusion, an improved method for the cleavage of conjugates in urine was developed using immobilized GRD and ARS packed into columns. This method combined the specificity of enzymatic hydrolysis with the speed of acid hydrolysis, leading to a fast, gentle cleavage. The production of the immobilizate columns consisted of an enzyme purification step followed by immobilization and column packing. The purified enzyme solutions and the immobilized enzymes could be stored for months without relevant loss in enzyme activities. The hydrolysis yields of NPG, NPS, and morphine conjugates in urine were sufficiently reproducible for a single column as well as for different columns even from different batches. The immobilizate columns could be reused at least 70 times without loss in the hydrolysis yields of morphine conjugates. Analyte carryover could be excluded by introducing a simple rinsing step with 200 mL/L methanol in standard acetate buffer. For cleavage of urinary conjugates using column-packed immobilized GRD and ARS, an incubation time of 1 h is recommended. Thus, this procedure was faster than using common enzymatic hydrolysis procedures because the applied enzyme activity was up to 45-fold higher. A study on the applicability in STA showed that conjugates of different drugs could be sufficiently cleaved in different authentic urines. Because the immobilizate columns could be reused, the costs for one hydrolysis were comparable to those of common procedures. Future studies will show the applicability of these immobilizate columns for automated sample preparation.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionDetermination of free morphine....References |
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- and ß-naphthols catalyzed by immobilized sulfatase. Biotechnol Bioeng 1983;25:919-928.
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