Published: Journal of Analytical Toxicology, Volume 24, Number 1, Jan/Feb, pp.70-72

Saliva/Plasma Ratio of Methadone and EDDP

To the Editor:

Recently, the interest in the use of saliva as an alternative medium to therapeutic monitoring has increased because it is a sample that offers several advantages: it is a noninvasive and painless method of sampling, it is readily available, and it does not require specially trained personnel. Its usefulness as an indicator of the clinical state depends on knowing the saliva/plasma concentration ratio for the drug studied, and this ratio has already been established for many drugs (1).

For methadone, some studies about saliva concentration were performed, but the results on the saliva/plasma ratio were contradictory. Some authors have studied the relationship between saliva and blood (2,3), but according to Plummer et al. (4), the determination of methadone in blood is influenced by the hematocrit of the patient because this drug is partially excluded from the red blood cells, and therefore this sample is not adequate for this analysis. Other authors (5–7) studied the saliva/plasma ratio (S/P): El-Guebaly et al. (5) found a S/P ratio of 4, Kang and Abbott (6) found a ratio of 0.51, and Wolff et al. (7) found a ratio of 1.3. However, none of these authors measured the salivary pH despite its importance to the salivary methadone concentration.

In the present work, we simultaneously determined methadone and 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), its main metabolite, in saliva samples from 10 patients in a methadone-maintenance program at Galicia Autonomic Community (Spain). We also evaluated the applicability of Salivette® (Sarstedt, Nümbrecht, Germany), a commercially available device for whole saliva collection, for the determination of these analytes.

Both saliva and blood were collected just before the administration of the daily dose of methadone and at least 30 min after consumption of any food or drink. The unstimulated saliva was collected by the spitting method. The patients were asked to close their lips and collect the saliva for a period of over 5 min, then spit it in a universal plastic container. Blood obtained by venipuncture was collected into heparinized tubes, and the plasma was separated by centrifugation for 5 min (5000 rpm) and transferred to polypropylene tubes. These samples were stored at –20°C until analysis.

Saliva and plasma samples were solid-phase microextracted (SPME) and quantitated by a gas chromatography–mass spectrometry (GC–MS) method previously described (8,9). The deuterated analytes (methadone-d₃ hydrochloride, Sigma Chemical Co. and EDDP-d₃ perchlorate, Radian Corp.) were used as respective internal standards. The analyses were performed on a Hewlett Packard model 6890 GC, equipped with an HP-5 capillary column (12 m x 0.22-mm i.d., 0.33-µm film thickness of crosslinked 5% phenyl methyl silicone). The injection port (splitless) was set at 250°C and the purge time was of 2.0 min. The column temperature was initially held at 90°C for 2 min, increased to 200°C at 30°C/min, held at 200°C for 5 min, then increased to 290°C at 30°C/min.

A Hewlett Packard model 5973 mass selective detector in selected ion monitoring (SIM) mode coupled to the GC was used for quantitative analysis. The electron impact of 70 eV was used for the ionization of the compounds, and the quantitation was based on target peak (values are given in parentheses) area ratios of methadone and EDDP with their respective internal standards. Ion currents at m/z (294), 295, and 223; (297) and 226; (277), 276, and 262; and (280), 279, and 265 were monitored for methadone, methadone-d₃, EDDP, and EDDP-d3, respectively.

Table I. Methadone and EDDP Concentrations in Plasma and Saliva of Maintenance Program’s Patients Subject no. Salivary pH Methadone dose (mg/day) Methadone dose (mg/kg/day) Methadone (µg/mL) EDDP (µg/mL) saliva plasma S/P* saliva plasma S/P* 1 5.0 50 0.73 1.30 0.30 4.3 0.07 0.04 1.8 2 5.0 125 1.47 2.72 0.38 7.2 0.05 0.03 1.7 3 6.0 80 1.01 0.68 0.35 1.9 0.09 0.05 1.8 4 7.0 80 1.45 0.12 0.21 0.6 0.09 0.17 0.5 5 7.0 350 4.49 1.60 0.40 4.0 0.09 0.15 0.6 6 5.0 250 3.52 2.58 0.48 5.4 0.10 0.11 0.9 7 6.0 70 0.88 1.24 0.32 3.9 0.04 0.10 0.4 8 5.0 100 1.38 1.20 0.46 2.6 0.04 0.07 0.6 9 5.0 150 2.31 3.46 0.60 5.8 0.07 0.42 0.2 10 6.0 160 1.95 1.06 0.64 1.7 0.04 0.11 0.4 * S/P : plasma/saliva concentration ratio.

Because the salivary pH may influence the concentration of methadone in saliva, we also measured this parameter in a pH meter (Basic 20 – Crison). Table I presents the concentration of methadone and EDDP found in the samples, the dose of methadone of each patient, the salivary pH, and the saliva/plasma ratio for both analytes. We found salivary concentrations ranging from 0.12 to 3.46 mg/L for methadone and from 0.04 to 0.1 mg/L for EDDP. In plasma, the concentrations found ranged from 0.21 to 0.64 mg/L for methadone and from 0.03 to 0.42 mg/L for EDDP. We found methadone S/P ratio values ranging from 0.6 to 7.2.

The correlation analysis shows a significant inverse correlation between salivary pH and methadone concentration (Figure 1). This is explained by the physicochemical properties of methadone and by the theory of diffusion. Methadone is a basic drug with pKa 8.2, and it is largely ionized in the unstimulated salivary pH (5.6–7). Thus, in normal conditions, the saliva is more acidic than blood, and because only un-ionized molecules diffuse through lipid membranes, methadone tends to accumulate in the saliva. For EDDP, the same analysis revealed that there is not a significant correlation between salivary concentration and pH, indicating that the salivary pH does not influence that parameter.

Wolff et al. (7) found a linear relationship between plasmatic and salivary concentrations and between dose (milligram per kilogram per day) and salivary concentrations studying 21 patients over a period of 30 months. We have not found these relationships, but the reduced members of participants in our study, 10 patients over a period of 12 months, could have influenced these results (Figure 2).

We also studied the applicability of Salivette, a commercially available device for saliva collection, to the determination of methadone and EDDP in saliva because this device could alter the quantitation of some substances (10). The Salivette consists of a conic tube with a suspended insert containing a cotton-wool swab (such as those used for dentistry) that, after being soaked with saliva, is reinserted, and the device is closed. After centrifugation, clean saliva is obtained.

We checked the recovery of the two analytes by comparing their concentrations in three control samples before and after the passage through this cotton wool swab. We found that there was a loss of about 40% for methadone and 30% for EDDP with Salivette compared to the direct measure. In addition, when we applied the device to real samples, the concentrations found were much lower than those obtained with the spitting method of collection, despite the mathematic correction, meaning that another factor may be influencing the results.

According to Kidwell et al. (11), putting cotton wool in the mouth is a way to stimulate salivary secretion, but this stimulation also leads to an increase in bicarbonate secretion and a concomitant rise in pH that could reduce the concentration of methadone in saliva as previously commented. In addition, the contribution of the parotid gland is increased to 50% of the saliva produced under stimulated conditions. This circumstance could have also contributed to the low concentration of methadone in real samples because methadone is not secreted by this gland in rats and human beings (12). Therefore, the Salivette seems to be inadequate for saliva collection when the purpose is the determination of methadone.

A.M. Bermejo
Institute of Legal Medicine, University of Santiago de Compostela, Spain

A.C.S. Lucas
Department of Clinical Analysis and Toxicology, University of Amazonas, Brazil, Rua Alexandre Amorim 330, 69010-300, Manaus-AM-Brazil

M.J. Tabernero
Institute of Legal Medicine, University of Santiago de Compostela, Spain

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