SpotSamplesas Determined by

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mazepine concentrations by enzyme multi- plied immunoassay: Interference with the. 10,11-epoxide metabolite ..... homogeneous enzyme immunoassay. (EMIT;.

4. Contin M, liva R, Albani F, et al. Determination of total and free plasma carbamazepine concentrations by enzyme multiplied immunoassay: Interference with the 10,11-epoxide metabolite. Titer Drug Monit (1984). In press,

Roberto Riva Manuela Contin Fiorenzo Albani Agostino Baruzzi Lab. of Neuropharrnacol., Neurology Univ. of Bologna Via U. Foscolo 7



40123 Bologna, Italy Giuseppe Dept. of Neurol. Giovanni XXIII Ban, Italy


Lamontanara Hosp.

Table 1. Correlationbetween TheophyllIne Results for Plasmaand BloodSpot Samplesas Determined by VariousAnalyticalMethods Errorof

Sampleand method compared x-axls Plasma, HPLC (3) Plasma, Optimate DBS, Fluorostat

n y.axIe Plasma, Optimate 50 DBS, Optimate 45 DBS, Optimate 40

approach that of the serum assay we decreased the volume of reagent used to elute the DBS disc (5’-sulfosalicylic acid, 50 g/L) to 270 iL and, after elution, combined 200 L of the eluate with 200 pL of the manufacturer’s concentrated BICINE assay buffer. This modification resulted in a reaction mixture having a pH of 8.20 (SD 0.017, n = 15) and produced the 51-fold dilution of patient’s sample required for the assay (the 6-mm DBS disc of PKU31 paper, now distributed by Whatman

improved Substrate-Labeled

FluorescentImmunoassayfor Theophyillne

in Dried-Blood


on Filter Paper

Ltd., contains 11.0 L of whole blood). The relationship between theophylline in DBS (y) and serum samples (x) by this modified procedure remained highly significant (n = 30, r = 0.997 1, y 0.8547, x = 0.08), and the slope was nearer to our original result. The pH difference in the reaction mixture between the serum and DBS assays had also existed in our original procedure =

To the Editor:

We previously described a substratelabeled fluorescent immunoassay for measuring the theophylline concentration in ifiter paper dried-blood spots (DBS), suitable for at-home therapeutic drug monitoring, based on use of the Ames/Miles “Fluorostat” instrument, which involved manual additions of reagents (Ames TDA) and polyclonal antibodies (1). Recently, the


of an automated and profluorometer (the Ames/Gilford “Optimate,” which more than halves TDA reagent consumption) and monoclonal TDA reagents has coincided with a change in the filter paper available for the assay. We therefore studied the effect of these changes on the original procedure and report a modified method suitable for the assay on both the manual and the automated analyzers. Capillary DBS (y) and plasma samples (x), collected from the same patient at the same time, were assayed grammable

for theophylline by using the Fluorostat analyzer, monoclonal TDA reagents, and the original protocol (1).

The relationship between the two sets of results (n = 38) was highly significant (r 0.9755, y = 0.9127x + 0.28), but the slope was 0.13 greater than that established for assays with the polyclonal antibody. =

To explain this difference, we investigated the pH of the assay reaction

mixtures, and found the DBS mixture to be significantly less basic (pH 7.90, SD 0.018, n = 20) than the plasma mixture (pH 8.27, SD 0.017, n 15). To have the pH in the DBS assay =

involving the polyclonal reagents but apparently had had no effect. We sus-

pect the monoclonal reagents, unlike the polyclonal, are pH sensitive but cannot confirm this because the polyclonal reagents are no longer available. To automate the DBS procedure for the Ames/Gilford Optimate we programmed the analyzer to “re-use the plasma dilution” (option 16 of the theophylline assay program). The diluted eluate (obtained as described above for

the modified Fluorostat procedure) is placed in the dilution cup, and the assay is completed by proceeding as pre-programmed by the manufacturer for the plasma assay. The relationships obtained (2) validated the Optimate results (Table 1). The therapeutic range for serum theophylline in asthma patients is typically 10 to 20 mg/L: the equivalent therapeutic range for capillary DBS is 8.6 to 17.1 mg/L. Other laboratories should determine their own relationship for serum/DBS values, which will depend on the characteristics of their particular punch and batch of filter paper. DBS assay reproducibility was adequate for clinical purposes. The modified Fluorostat procedure gave withinbatch CVs (n = 10) of 4.6% and 4.3% and between-batch CVs (n = 10) of 7.1% and 6.9% for DBS theophylline concentrations of 9.2 and 19.8 mg/L, respectively. The Optimate procedure gave within-batch CVs (n = 10) of

RegressIonequatIon y=0.9607x + 0.071 y=0.8200x + 0.03 y=0.9543x + 0.073


0.9950 0.9955 0.9958

regressIon about y 0.4801 0.3921 0.3287

5.1%, 6.6%, and 5.2% for DBS theophylline concentrations of 6.9, 11.9, and 18.8 mg/L, respectively, and between-batch CVs (n = 12) of 6.9% and 5.8% for DBS theophylline concentrations of 11.7 and 23.4 mg/L, respectively.



to the clinician


capillary DBS samples that can be collected and mailed from the patient’s home to the laboratory for the therapeutic drug monitoring of theousing

phylline and phenytoin in children, adults, and the elderly have been described elsewhere (1, 4-6). The automated Optimate analyzer offers the laboratory many advantages. Reagent

and buffer additions as well as fluorescence measurements areautomatically performed and timed, decreasing operator involvement and hence assay cost. Fluorescence is read earlier-4.5 mm vs 20 mm on the Fluorostat-thereby shortening assay time. Nonspecific fluorescence is also minimized. Consumption of antibody/enzyme and fluorogenic drug reagent is decreased from 50 1LLto 20 L per test. This is important when determining profiles, in which duplicate samples typically are collected when peak and trough concentrations of a circulating drug are expected, or when the patient is likely to suffer symptoms (e.g., the “early morning dipper”). Storage of standard-curve data (typically for at least three to four weeks) alsodecreases reagent consumption be-

cause most assay batches will


consist of quality-control and patients’ samples only. The Optimate analyzer has so far performed reliably, has been

well accepted by laboratory personnel, and thus seems well suited to monitor theophylline therapy with either conventional


spots of capillary


or dried


References 1. CoombesEJ, Gamlen TR, Batstone GF, Holgate ST. The validation of a fluoroimmunoassay for the determination of theophylline concentration in dried blood spots suitable for domiciliary therapeutic drug monitoring. Clin Chim Acta 136, 187-195 (1984).

2. Cornbleet

PJ, Gochman N. Incorrect least squares regression coefficients in method comparisonanalysis. Ciin 25, 432-438 (1979).


CLINICAL CHEMISTRY, Vol. 31, No. 1, 1985


3. James HC, Garden TR, Batstone GF. An appraisal of a fluoroimmunoassay technique for the determination of blood theophylline concentration.Ann Clin Bicchem 20, 251-253 (1983). 4. Coombes LI, Garnlen TR, Leigh PN, BatstoneGF. A phenytoinassay using dried bloodspot samples suitable for domiciliary therapeuticdrug monitoring.Ann Clin Biochem (in press). 5. Batstone GF, CoombesLI, Gamlen TR. Theophylline assay on dried blood spots.

Lancet ii,109-110 (1983).Letter.

6. Batstone GF, CoombesLI, Garden TR. Measuring theophylline on dried blood spots. Lancet ii, 99(1984). Letter. E. J. Coombes G. F. Batstone Dept. of Chem. Pat/wi. Salisbury Infirmary Salisbury, Wilts. SP2 7SX, UJC.

T. R. Gamlen T. Stickland Dept. of Chem. Pat/wi. Milton Milton

Keynes Keynes,


Hospital UJC.

MK6 5W,

for BenzodiazepinesIn Urineafter Hydrolysisof GlucuronideConjugates Screening

To the Editor: The EMIT Urine Benzodiazepine Assay (Syva Diagnostics, Palo Alto, CA) is marketed for the detection of benzodiazepine misuse. The recommended limit of reliable detection with 95% confidence has been variously stated as 0.3 or 0.5 mg/L with oxazepam as a reference. When we obtained several falsely negative results for random urine samples from patients in this hospital who had received 30-mg doses of oxazepam the previous night, we conjectured that most of the drug was excreted as corjugated metabolites, which were not being detected by the test system. In investigating this possibility, we found that the sensitivity of the method could be markedly increased by incubating urine samples with f3-glucuronidase (/3D-glucuronide glucuronylhydrolase; EC from Helix pomatia (“crude solution”; Sigma Chemical Co., St. Leuis, MO). Urine samples collected over 36 h after a patient had completed a twoday course of oxazepam (30 mg, twice daily) were treated by adding 0.05 mL of crude enzyme (as supplied) to 3.0 mL of urine and incubating at ambient temperature for 2 h. Absorbance was

read at 340 nm according to the recommended procedure, after a delay time of 30 s and again 60 s later. The changes in absorbance were as follows:

metabolite.With lots M02 and M03 of this assay, greater than 500 jig of oxazepam glucuronide per milliliter would have to be present to be detected. After oxazepam administration, the metabolite composition in urine consistsalmost entirely of oxazepam glueuronide (1). Nevertheless, the authors Before After Urinesample hydrolysis hydrolysis cite negative responsesfor only some of the urine samples from persons given Pro-dose 681 675 30-mg doses of oxazepam; this indiPost-dose 6 h 817 1557 cates that trace amounts of other 12 h 1321 882 metabolites must be contributing to 24 h 707 1252 36 h 867 1144 the positive test responses. Oxazepam 890 867 With other ben.zodiazepines, addicalibrator, tional metabolic pathways produce de0.3 mg/L methylated or hydroxylated urinary metabolites, or both. These metabolites The recommendations of the manuare also highly conjugated, but less facturer direct that, in general, #{163}4extensively than oxazepam. For invalues should exceed those obtained stance,approximately 53% of a dose of with the calibrator before a positive diazepam was present in urine as gluresultcan be reported with 95% conficuronide conjugates (2). The unconjudence. By this criterion, the present gated metabolites have high reactivity results show that patients on a “therain the assay. Therefore, this assay may peutic regime” of oxazepam are likely be relatively less sensitive in detecting to show negative results, unless the the usage of oxazepam, but it is highly urinary drug conjugates are first hysensitive in detecting the usage of drolyzed. After hydrolysis, the theramany other benzodiazepines without peutic use of oxazepam is detectable for the necessity of hydrolysis. In all cases, at least 36 h after the last dose. the assay is intended to detect abusive The EMIT benzodiazepine assay, as use of certain drugs, not to detect very marketed, will sometimes give a posilow therapeutic doses of benzodiazetive result after a 10-mg dose of diazepines. pam; however, after a 5-mg dose the References drug is not detectable in urine (1). After the enzyme treatment step rec1. Sisenwine SF, et al. Arzneim Forsch 22, ommended here, a single 5-mg dose of 682 (1972). diazepam was detectable in a urine 2. Baselt RC. Diazepam. In Disposition of sample collected 24 h after the dose. In Toxic Drugs and Chemicals in Man, 1, some circumstances, a change in the Biomedical Publications, Canton, CT post-hydrolysis #{163}4 value (relative to 06019, 1978, p 128. the pre-hydrolysis value) may be a Joyce Chang more sensitive indicator of benzodiazepine use than comparison with a Syva Co. calibrator of unknown relevance-a P.O. Box 10058 possibility we are investigating furPalo Alto, CA 94303 ther.

Reference 1. Turner J, Chang J. EMIT assaysfor drugs of abusein urine, TDM-Toxicology Continuing Education Program, Am. Assoc. for Clin. Chem.,Washington, DC, 1983.

P. T. Mearrick

J. S. Chahl Depts. of din. Pharmacol. and Clin. Chem. Royal Newcastle Hosp.



TDx Procedure



A Syva representative


To the Editor: Recent studies conducted by Syva on the reactivity of the EMIT#{174} d.a.u. Benzodiazepine Assay to oxazepam glueuronide indicate a low reactivity to this

152 CLINICALCHEMISTRY, Vol. 31, No. 1, 1985

Influence of Uremiaon Four Assays for Theophylline: Improved Results with a MonocionaiAntibodyin the

To the Editor: Recent reports indicate that the fluorescence polarization immunoassay for theophylline in the TDx (Abbott Laboratories, North Chicago, IL) gives higher values for serum from patients with chronic renal failure than does homogeneous enzyme immunoassay (EMIT; Syva Co., Palo Alto, CA) (1,2) or “high-performance” liquid chromatography (HPLC) Here we report confirmation of these observations