Heat stabilization of blood spot samples for determination of metabolically unstable drug compounds

Abstract

Background

Sample stability is critical for accurate analysis of drug compounds in biosamples. The use of additives to eradicate the enzymatic activity causing loss of these analytes has its limitations.

Results

A novel technique for sample stabilization by rapid, high-temperature heating was used. The stability of six commercial drugs in blood and blood spots was investigated under various conditions with or without heat stabilization at 95°C. Oseltamivir, cefotaxime and ribavirin were successfully stabilized by heating whereas significant losses were seen in unheated samples. Amodiaquine was stable with and without heating. Artemether and dihydroartemisinin were found to be very heat sensitive and began to decompose even at 60°C.

Conclusion

Heat stabilization is a viable technique to maintain analytes in blood spot samples, without the use of chemical additives, by stopping the enzymatic activity that causes sample degradation.

Sample stability during sampling, transport, storage, sample preparation and analysis is crucial to ensure accurate analytical results. Method validation has to take into account the target analyte stability. Instability of sample components other than the target analytes may also affect the measurement, so stability of the sample matrix components and of compound structures that are closely related to the target analyte (e.g., glucuronides) are of interest. In the case of metabolite profiling, sample stability includes, more or less, the entire sample and is not limited to specific components [1]. Sample instability is often, but not solely, due to enzymatic activity [2]. Activities in plasma and at the surface and in the interior of blood cells include, for example, ester hydrolysis, deamidation, dealkylation/hydroxylation and phosphorylation [3,4]. Stabilization procedures can be designed to aim at specific targets. For example, more- or less-specific inhibitors have been used to stop protease or esterase activity [4]. On the other hand, chemical additives with nonspecific inhibitory action have been used, for example, sodium dodecyl sulphate [5,6]. In these cases, high concentrations of the additives are often needed, which may interfere with the determination of analytes [7]. Blood sampling with dichlorvos or fluoride/oxalate has been used to prevent ester hydrolysis of oseltamivir [8,9]. However, it has been claimed that inhibitor cocktails and other additives do not ensure eradication of all enzymatic activity in tissue samples [10].

The use of dried blood spots (DBS) involves drying the sample, a step that has been shown to prevent degradation [11]. Sample degradation can be counteracted by keeping the time span short between blood collection and spotting, and by keeping the collected blood on ice prior to spotting. However, after spotting, the spot is left to dry at ambient temperature and it may take up to 2 h, possibly more, before drying is complete. For substances with half-lives shorter than several hours in fresh blood, this standard DBS procedure may not be adequate: there is a risk that the sample composition has been altered before the components have been stabilized by drying [12].

Heating samples for a short period of time at a precise temperature aims to stop all enzymatic activity by denaturing the proteins responsible for enzymatic activity and does not involve chemical additives. This heating technique has been proven to be effective for stabilizing tissue samples [13,14]. In this work, heat stabilization has been applied to blood microsamples, either as blood spots on cellulose or glass fiber paper, or as blood drops placed on a thin foil.

Six different drug compounds (Figure 1) have been included in the tests. For five of these instability in blood and/or plasma has been reported: oseltamivir [15], artemether and dihydroartemisinin [16], ribavirin [17] and cefotaxime [18]. Oseltamivir is an ethyl ester that is converted by esterases to its active metabolite the free carboxylic acid. Ribavirin may be subjected to phosphorylation. Artemether and dihydroartemisinin are peroxides with limited chemical stability in blood; degradation mechanisms have not been fully elucidated. Cefotaxime is subjected to degradation in contact to red blood cell components. Amodiaquine was included in the tests as a drug with reported stability in blood spotted on paper [19].

Figure 1. Model compounds used for stabilization experiments.

Initial tests were made measuring butyryl esterase activity measurements, in order to find temperature and duration settings for heat stabilization, which could be used as default conditions in the other experiments.

Experimental

Materials

Oseltamivir, oseltamivir carboxylate (OSC) and their tri deuterated internal standards (IS) were obtained from F. Hoffmann-La Roche Ltd. (Basel, Switzerland). Ribavirin was obtained from Sigma-Aldrich (MI, USA). Dihydroartemisinin was obtained from Guangzhou University of Traditional Chinese Medicine (Guangzhou, China). Artemether and the stable isotope-labeled IS artemether-¹³CD₄ and dihydroartemisinin-¹³CD₄ were obtained from Novartis (Basel, Switzerland). Amodiaquine was obtained from ALSACHIM (Illkirch Graffenstaden, France) and its IS from Sterling-Weintorph (Hertfordshire, UK). Cefotaxime was obtained from Santa Cruz Biotechnology, Inc. (CA, USA). For esterase activity measurements, 5,5-dithio-bis-2-nitrobenzoic acid (Fluka) and S-butyrylthiocholine iodide (Fluka) were purchased from Sigma-Aldrich (Schnelldorf, Germany). Phosphate buffer, pH 8.0 were prepared by mixing disodium hydrogen phosphate (Fluka), hydrochloric acid (Sigma) and Milli-Q water also purchased from Sigma-Aldrich.

Filter papers used were Whatman ET31 Chr and Whatman FTA DMPK-C from Whatman International (Maidstone, UK) and Agilent Bond Elut DMS card from Agilent Technologies (CA, USA).

Water, acetonitrile (ACN) and methanol were obtained from JT Baker (NJ, USA). Formic acid (98–100%), acetic acid, ammonium acetate (LC–MS grade), potassium dichromate and deferoxamine mesylate were from Sigma-Aldrich. Sodium hydroxide (pellets) and tri ethylamine were obtained from BDH (Poole, UK). Ammonia (25%), ortho-phosphoric acid (85%) and perchloric acid (70–72%) were obtained from Merck (Darmstadt, Germany).

Heat stabilization was performed using a Stabilizor™ instrument from Denator (Gothenburg, Sweden). Whole-blood samples were heated in Maintainor™ cards.

Methods

Preparation & heat treatment of blood samples

Working standard solutions of drug compounds were prepared in a 1:1 mixture of human plasma and water, and used to spike whole blood (kept on an ice bath) with no more than 5% dilution of the blood. Blood samples were spotted onto filter paper, using an Eppendorf pipette, shortly after spiking. Blood spots were placed in Maintainor cards and processed in the Stabilizor instrument within 20 s after spotting or after a scheduled time period. Time points for sample treatment were logged automatically by sthe Stabilizor instrument. Unless stated otherwise, heat treatment was carried out for 30 s with instrument temperature set at 95°C, and the blood spot sample was allowed to dry in open air at ambient temperature. Samples from experiments carried out in a laboratory in Sweden with oseltamivir and artemether/dihydroartemisinin were packed in plastic bags containing silica gel and sent by air transport for analysis in Thailand. No results from analysis on day 0 were obtained from such samples.

Determination of butyrylcholinesterase activity

Activity of blood spot extracts was measured using the method of White et al. for plasma samples, with minor modifications [20]. Two 3-mm discs (Harris Uni-Core Punch) from a spot, corresponding to 8 μl of blood, and 200 μl of 0.1 M phosphate buffer, pH 8.0, were shaken for 30 min. A 100 μl aliquot of the extract was placed in a 96-well flat bottom plate (Costar, Corning Inc., NY, USA) and mixed with 10 mM S-butyrylthiocholine iodide (80 μl) and 10 mM 5,5-dithio-bis-2-nitrobenzoic acid solution (20 μl). Absorbance at 412 nm was measured directly and after 5, 10, 15 and 20 min using an Infinite M200 microplate reader (Tecan, Basel, Switzerland). Butyrylcholinesterase activity α was calculated using the following formula:

$$\alpha =(\Delta \mathrm{A}∕\Delta \mathrm{t})\times {10}^6\times (\mathrm{DF}∕\epsilon )$$

where α is the activity (μmol × l⁻¹ × min⁻¹), ΔA/Δt is the rate of change in absorbance (min⁻¹), DF is the dilution factor for the blood sample and ε is the extinction coefficient for dinitrobenzoate (13600 mol⁻¹).

Determination of drug compounds

Oseltamivir: a method for plasma samples was slightly modified for DBS [21]. Briefly, four discs of 3.2 mm were punched from each DBS, placed in a 1-ml 96-well plate and extracted by adding 50 μl of IS and 600 μl ammonium acetate 5 mM, pH 3.5, and mixed for 10 min. After centrifugation, 500 μl were transferred to a MPC-SD standard 96-well SPE plate (3M Empore, Bracknell, UK) and extracted.

Artemether and dihydroartemisinin: a method was modified for DBS [16]. Briefly, seven discs of 3.2 mm were punched from each DBS and extracted by adding 10 μl of potassium dichromate 0.08 M and 10 μl deferoxamine 20 mg/ml as stabilizers. IS (550 μl in ACN) was added followed by mixing for 60 min. After centrifugation, 450 μl was transferred to a new 96-well plate and the solvent was evaporated under a gentle stream of nitrogen. Samples were reconstituted in 150 μl of mobile phase solvent.

Amodiaquine: samples were prepared according to a scaled-down version of an existing DBS method designed for 100 μl blood spots [22]. Three discs of 3.2 mm were punched from each DBS and extracted by adding 200 μl of 0.3 M perchloric acid and mixed for 60 min followed by addition of 100 μl of ACN and mixing for 10 min. IS (500 μl in phosphate buffer, pH 2) was added before transferring all sample to an PRS (strong cation exchange) SPE column (Biotage, Uppsala, Sweden). Methanol–triethylamine (98/2 v/v) was used for elution. After solvent evaporation, the sample was reconstituted in 400 μl of mobile phase liquid (ACN/ammonium formate 20 mM with 1% formic acid 8/92 v/v).

Ribavirin and cefotaxime: a fast and simple procedure was used without IS. Three discs of 3.2 mm were punched from each DBS and 300 μl of extraction solvent was added (for ribavirin:ACN/buffer [10 mM ammonium acetate + 1% acetic acid] 10/90 v/v and for cefotaxime:ACN/formic acid 0.5% 20/80 v/v) and mixed for 30 min. The extract was then transferred to a new plate and diluted with 300 μl (ACN for ribavirin and mobile phase solvent for cefotaxime, respectively).

Pretreated samples were separated on a Model 1200 system consisting of a binary LC pump, vacuum degasser, temperature-controlled micro-well plate autosampler set at 4°C (20°C for oseltamivir/OSC) and a temperature-controlled column compartment set at 40°C (20°C for oseltamivir/OSC) (Agilent Technologies). Injection volume was between 1 and 5 μl. A flow rate of 500 μl/min was used for all methods. oseltamivir and OSC were separated on a ZIC®-HILIC column (50 × 2.1 mm; Sequant, Umea, Sweden), using an ACN/ammonium acetate buffer (pH 3.5; 10 mM) gradient. For ribavirin, the same chromatographic system was used with a different gradient. Artemether and dihydroartemisinin were separated on a Hypersil™ Gold C18 100 × 2.1 mm ID, 5 μm (Thermo Electron Corporation, MA, USA) using a mobile phase of methanol/ammonium acetate buffer (pH 3.5; 10 mM) 70/30 v/v. Amodiaquine was separated on a Halo C18 50 × 2.1 mm ID, 2.7 μm column (Advanced Materials Technology, DE, USA) with a mobile phase of ACN/ammonium formate 20 mM with 1% formic acid 8/92 v/v. Cefotaxime was analyzed on a Zorbax SB CN column, 50 × 4.6 mm ID, 3.5 μm (Zorbax Inc., NC, USA) using a mobile phase of ACN/formic acid 0.5% 30/70 v/v. Analytes were detected by ESI–MS using an API 5000 or an API 3200 triple-quadrupole mass spectrometer with TurboV™ ion source (AB Sciex, Concord, ON, Canada). Acquisition and data processing was performed using Analyst® 1.5.2 software. Mass transitions for selected reaction monitoring and parameter settings are listed in Table 1.

Table 1. Instrument and settings for LC–MS/MS analysis.

Analyte	MS system	Mass transition (m/z)	Ion spray voltage (V)	Drying gas temperature (°C)	Curtain gas N2 (psi)	Nebulizer gas (GS1) N2 (psi)	Heater gas (GS2) N2 (psi)
Oseltamivir	API 5500	313.3→225.1	5500	575	30	50	45
Oseltamivir carboxylate	API 5500	285.2→197.1	5500	575	30	50	45
Artemether	API 5500	316.25→163.15	4500	300	25	55	60
Dihydroartemisinin	API 5500	267.2→249	4500	300	25	55	60
Ribavirin	API 3200	245.3→113.1	5500	500	30	60	60
Amodiaquine	API 3200	356→283	5500	650	25	60	60
Cefotaxime	API 3200	454.1→238.9	−4500	400	25	55	55

Results & discussion

Butyrylcholinesterase activity

Human Li–heparin blood (40 μl) was spotted (n = 2) on Whatman ET31 Chr paper and either heated for 15, 30 or 45 s at instrument-set temperatures of 90, 95 or 98°C, or left untreated. After drying, 3-mm discs of blood spots were extracted with 0.1 M phosphate buffer, pH 8.0, and butyrylcholinesterase activity of the extracts was measured. With a temperature setting of 90°C, activity remained after a 15 s treatment, but was eliminated by a 30 s treatment. An instrument setting of 95 or 98°C, 15 s was sufficient to eradicate butyrylcholinesterase activity (Table 2).

Table 2. Butyrylcholinesterase activity (nmol × ml⁻¹ × min⁻¹) of blood spot samples after heat treatment with Stabilizor™ instrument.

Time (s)	Temperature (°C)
	90	95	98
0	125	129	139
15	121	1	0
30	0	1	0
45	−1	1	−1

Drying of blood spots

Filter paper was weighed before spotting and at intervals during the course of drying. Heat treatment for 30 s at 95°C (nominal temperature) resulted in an initial loss of weight of 10–15%. The following drying profile was similar with or without heat treatment, and after 40 min no significant differences were exhibited, which means that the time required for complete drying was the same with or without heat treatment. The spot weight after 1 h was 20% of the weight directly after spotting (Figure 2).

Figure 2. Net weight of a 25-μl blood spot after spotting and up to 65-min drying, with or without initial heat treatment (30 s at 95°C instrument set temperature).

Weight at time 0 was set to 100%.

It was also observed that heat-treated blood spot samples did not smear and that samples could be put in plastic bags with silica desiccant without previous drying. The sample is then allowed to dry in the closed bag. The drying process, although slower than in the open air in a dry environment, will be independent of the humidity in the environment.

Oseltamivir

Human K₂-EDTA blood (pooled from three individuals) and mouse blood (pooled from two individuals) was spiked with oseltamivir at 2000 ng/ml (6.4 μmol/l). Blood (25 μl) was either spotted on Whatman DMPK-C cards and left to dry, or spotted onto filter paper cut from the cards and heated directly after spotting before being left to dry at room temperature. Blood (25 μl) was also placed as a drop and left to dry on foil. Samples were shipped and stored at ambient temperature until analysis 10 days later for content of oseltamivir and oseltamivir carboxylate. No formation of the carboxylate was observed in human blood samples due to lower esterase activity than in rodent blood irrespective of treatment. Formation of the carboxylate was observed in mouse blood samples: 81 ± 3% (n = 3) conversion was seen in the 25 μl blood drops left to dry in ambient air, while 26 ± 3% (n = 6) was seen for mouse blood spotted onto cards without heat treatment. For blood that was spotted and heated (30 s at 95°C), 2.3–4.8% conversion was seen (Figure 3). When percentage conversion was plotted against time between spiking and heat treatment, a time dependence was seen clearly, indicating that all or most of the conversion occurred prior to heat treatment. In another experiment, heat treatment of 30 or 60 μl of mouse blood for 15, 30 or 60 s was compared. No differences in conversion rate were observed except those that could be explained by differences in time between spiking the blood and spotting the sample.

Figure 3. Conversion of oseltamivir to oseltamivir acid.

(A) Percentage of oseltamivir converted to OSC (100 × [OSC]/[oseltamivir + OSC]) in mouse blood samples with different treatments. (B) Oseltamivir and OSC found in six replicates spotted in sequence and heat treated directly after spotting. (C) Percentage of oseltamivir converted to OSC for six replicates plotted against storage time (time elapsed between initial spiking of blood with oseltamivir and heat treatment of the spotted sample).

DBS: Dried blood spot; OSC: Oseltamivir carboxylate.

Ribavirin

Human blood (Na–heparin) was spiked with ribavirin at 10 μg/ml. Blood (40 μl) was spotted in triplicate on Whatman ET 31 Chr paper and allowed to dry at room temperature (~23°C, 50% relative humidity [RH]). One set of samples was allowed to dry for 0, 10, 30, 90 or 180 min, then heated and placed in a freezer at −80°C. A second set was heated directly after spotting, then left to dry for different time periods prior to freezing. For comparison, 40 μl of whole blood was placed as a liquid drop directly onto the foil of the Maintainor card, and treated as the first series of blood spot samples. Thawed samples were analyzed for their content of ribavirin as described in the ‘Experimental’ section. Recoveries were estimated using a neat standard solution as a reference. Results are shown in Figure 4. Consistently high recoveries were obtained when blood spots were heated directly after spotting. In these cases, no differences were seen between samples with different drying times prior to freezing. Blood spots that were left to dry untreated, and heated just before freezing, gave somewhat lower recoveries, with a trend to lower recoveries for longer drying times. Such a trend was more pronounced for whole blood left to dry as a drop on foil. In conclusion, ribavirin degradation was less in blood spots than in whole blood, but was still observable. The high recoveries of ribavirin from blood spots, heated directly after spotting indicated that not only had enzymatic degradation stopped, but also that analytical recoveries from heated spots were consistent and as high as recoveries from unheated spots.

Figure 4. Recovery (mean ± SD; n = 3) of ribavirin from blood spots and whole-blood samples, heat treated either immediately or after 10–180 min drying.

DBS: Dried blood spot.

Artemether & dihydroartemisinin

Blood (Na–heparin) was kept on ice and spiked with the two closely related compounds, artemether and dihydroartemisinin, both at 500 ng/ml. Samples were spotted in triplicate directly or mixed with 0.4 M potassium dichromate solution (4:1) on Whatman DMPK C cards. Blood spot samples, with or without 30 s of heating at 95°C, were left to dry at room temperature, shipped and stored for 7 days prior to analysis. Samples stabilized with potassium dichromate, but not heated showed recoveries of 75 ± 2% and 69 ± 2% for artemether and dihydroartemisinin, respectively. For samples that had been heated, recoveries were lower: 38 ± 13% and 38 ± 14% for artemether and dihydroartemisinin, respectively. Samples not stabilized with potassium dichromate and not heated showed recoveries of 49 ± 10% and 27 ± 6%, while samples that had been heated showed zero recovery of artemether and dihydroartemisinin. In a follow-up experiment, the heat sensitivity of the two compounds was further investigated by varying the conditions for heat treatment: 15, 30, 45 or 60 s, and 60 or 95°C (samples in triplicate). At 60°C, a time-dependent decrease was seen, with approximately 50% recovery for both artemether and dihydroartemisinin from the 60-s treatment. With heating at 95°C, total loss of both analytes was seen after 15 s (Figure 5).

Figure 5. Recovery (mean; n = 3) of artemether and dihydroartemisinin from blood spotted on Whatman DMPK C cards with 0–60 s heat treatment at eiher 60 or 95°C before drying.

Amodiaquine

Human blood (Na–heparin) was spiked with amodiaquine at 10 μg/ml and spotted onto filter paper. Spots were left to dry for at least 2 h at room temperature (approximately 23°C, 50% RH), without heating or after heating for 30 or 60 s, then left at room temperature for 0, 7 or 21 days and stored frozen at −80°C until analysis (triplicate samples). Samples were analyzed for amodiaquine content on one occasion. No significant differences in analyte recovery were seen between different heating times or between 0, 7 and 21 days of storage (Figure 6).

Figure 6. Recovery (mean analyte-to-internal standard peak area ratio; n = 3) of amodiaquine from blood spots stored for 0, 7 or 21 days at room temperature.

Blood spots were either heat treated for 30 or 60 s or untreated prior to drying for 2 h.

Cefotaxime

Human blood (Na–heparin) was spiked with cefotaxime at 10 μg/ml. Blood (40 μl) was spotted in triplicates on Whatman ET31 Chr paper and left to dry at room temperature (approximately 23°C, 50% RH) for 0, 10, 30, 90 or 180 min before placing samples in a freezer at −80°C. One series of samples (A) was not heated. A second series (B) was heated after the drying period, before freezing. A third series (C) was heated directly after spotting. In another experiment, a series of samples (D) of whole blood containing 5% hemolyzed blood and spiked with cefotaxime were placed onto foil and left to dry for 0–180 min prior to freezing. Thawed samples were analyzed for content of cefotaxime as described in the ‘Experimental’ section. Recoveries were estimated using a neat standard solution as reference. Results are shown in Figure 7.

Figure 7. Recovery (mean ± SD; n = 3) of cefotaxime from whole blood spotted on Whatman ET31 Chr paper or from whole blood (5% hemolyzed) spotted onto foil.

(A) Blood spot frozen after drying for 0–180 min, (B) blood spot heat treated after drying for 0–180 min then frozen, (C) blood spot heat treated directly after spotting, dried for 0–180 min and then frozen, and (D) blood (5% hemolyzed) on foil, dried for 0–180 min then, heat treated and frozen.

Samples from series A that had been dried for only 30 min or less, all showed recoveries of less than 10%, while drying for 60 or 180 min showed higher recoveries of approximately 40%. A plausible explanation is that the blood spot is still wet when put into the freezer; freezing causes hemolysis; the thawed sample, still wet, exhibits increased enzymatic activity resulting in loss of cefotaxime before the analyte is extracted and, thus, inactivated. Samples dried for 60 or 180 min are more or less dry and exhibit less or no enzymatic activity when thawed.

Samples from series B that had been dried to different time points then heated prior to freezing showed higher recoveries, compared with series A, when dried for 0–30 min, and similar recoveries from 30–180 min drying. There was a trend towards decreasing recoveries with longer times prior to heat treatment, which could be due to cefotaxime degradation during drying.

Samples from series C, heated directly after spotting, showed consistently high recoveries. A weak trend towards lower recoveries (from 70 to 66% after 0–180 min) was observed. Since blood was not spotted and treated in random order, but in order of increasing drying time, this could be due to cefotaxime degradation prior to spotting.

Recoveries of cefotaxime from hemolyzed whole-blood samples (series D) decreased rapidly with time, supporting the hypothesis that hemolysis contributed to the low recoveries observed for samples in series A, which had been frozen and thawed when still wet.

In conclusion, the experiments showed that heat treatment protected cefotaxime from degradation. Drying without heat treatment may result in loss of cefotaxime, which can be quite severe in partially hemolyzed samples.

Conclusion

Using a standard Stabilizor T1 instrument with heating temperature set at 95°C, enzymatic activity in blood was efficiently stopped by heating blood spot samples for 30 s. The drug compounds tested showed different degrees of instability in blood spots without heat treatment. With the exception of artemether and dihydroartemisinin, heat treatment successfully stabilized samples with no apparent difference in extractability of the analyte from the blood spot. Losses of artemether and dihydroartemisinin were seen already at a temperature of 60°C. The technology makes use of commercially available hardware and consumables and fits into preclinical and clinical workflows.

Future perspective

Stabilization by rapid heat treatment is a complementary technique that can be included in different sample handling protocols for biotissues and biofluids in order to stop enzymatic degradation. Applications may include proteins with post-translational modifications, endogenous peptides and low-molecular-weight drugs. There may be more applications where heat treatment could solve stability issues, whether it is the target analyte or other sample components that require stabilization. Drug compound instability due to enzymatic activity is not uncommon in preclinical (rodent) studies. In clinical studies, the unstable compound typically is a prodrug rather than the active drug compound. The future of DBS in clinical bioanalysis is dependent on eliminating or limiting the so-called hematocrit effect, that is, inaccuracy caused by hematocrit variability. Whether the solution will involve punching a disc or taking the whole spot for analysis, heat stabilization may, in both cases, be used in conjunction with blood spotting. The prospect of immediate packaging of heat-treated DBS samples together with silica gel for drying is to be evaluated in future studies and could be a reason to use heat treatment also when no stability issue has been identified.

Executive summary.

• Drying of blood spots, in terms of drying time, was not affected by the heat treatment.

• Butyrylcholinesterase activity was eliminated by a 30 s heat treatment at 95°C.

• Extensive conversion of oseltamivir to oseltamivir acid in mouse blood samples was effectively stopped by heat treatment.

• Ribavirin showed minor degradation during drying of blood spots, which was eliminated by heat treatment.

• Arthemether and dihydroartemisinin were degraded by heat treatment already at 60°C.

• Amodiaquine showed no degradation in blood spots during drying and analytical recoveries were the same for heat-treated and untreated blood spots.

• Degradation of cefotaxime in blood spot samples during drying was accelerated by blood hemolysis. Heat treatment effectively stopped degradation.

• Heat-treated blood spot samples did not smear, which allowed samples to be put in plastic bags with silica desiccant without previous drying. Stabilization by heat treatment eliminates the risk of matrix effects caused by adding chemicals to the sample for stabilization.

Key Terms

Analyte stability

Consistency of analyte concentration in the sample matrix over time.

Enzymatic activity

Property of proteins in the sample promoting chemical reactions.

Dried blood spots

Technique for handling small whole blood samples, typically 5–50 μl, on filter paper.

Heat stabilization

Heating the sample, typically for less that 1 min, to prevent enzymatic degradation of sample components.

Sample treatment

Actions carried out on samples in conjunction with and after sample collection and prior to instrumental analysis