The Chemical Stability and Sterility of Sodium Thiopental After

Preparation
Haws, J. Larry MD; Herman, Norman MD, PhD; Clark, Yoshimi RPh, BCNSP; Bjoraker, Robert MD; Jones, David PhD
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Author Information
Departments of Anesthesiology (Haws, Bjoraker) and Pharmacy (Clark), Witford Hall Medical Center Lackland Air
Force Base, Texas; Anesthesia Research Laboratories, Department of Anesthesiology, University of Texas Health
Science Center at San Antonio, San Antonio, Texas (Jones); and Department of Anesthesiology, New York
Hospital-Cornell Medical Center, New York, New York (Herman).
Financially supported by the Directorate of Education at WHMC, Department of the Air Force.
The views expressed in this article are those of the authors and do not reflect the official policy of the Department
of Defense or other Departments of the Government.
Portions of this study were presented at the Post Graduate Assembly of the New York State Society of
Anesthesiologists annual meeting in December 1995, New York, NY.
Accepted for publication September 18, 1997.
Address correspondence to J. Larry Haws, MD, Department of Anesthesiology/SGOSA-H, 77th Medical Group,
10535 Hospital Way, Mather Air Force Base, CA 95635.

Abstract
Manufacturer's instructions recommend discarding unused portions of sodium thiopental 24 h after
reconstitution.Heeding this recommendation may result in the disposal of a large proportion of prepared
thiopental. Although thiopental is relatively inexpensive, the volume prepared by many anesthesia departments
could make this waste significant. To address this possibility, we investigated the chemical stability and sterility of
thiopental in pharmacy-prepared, prefilled syringes. Stock solutions of thiopental were mixed and drawn into
syringes under sterile conditions by pharmacists or pharmacy assistants. Fifty-six samples were stored under
refrigeration (3[degree sign]C); the remaining 56 samples were stored at room temperature (22[degree sign]C).
Each day for 7 days, eight samples from each group were analyzed by using high-performance liquid
chromatography for chemical stability and cultured for microbiological colonization. Differences in thiopental
concentration between the room temperature and the refrigerated samples were measured over time by using
repeated-measures analysis of variance (P <or=to 0.05). Three positive culture samples (S. epidermidis and S.
hemolyticus) most likely represent laboratory contamination and not colonization. At 22[degree sign]C, thiopental
remains stable and sterile for 6 days and well beyond 7 days at 3[degree sign]C. Implications: This study
examines the shelf life of the anesthetic drug thiopental in pharmacy-filled syringes stored at either room
temperature or under refrigeration. The results justify the use of prepared solutions beyond the package insert
recommendation of 24 h.
The package directions for sodium thiopental (Pentothal[registered sign]) recommend that reconstituted solutions
should be "used promptly" and that "unused portions should be discarded after 24 hours" (package insert 6-4781R9-Rev., thiopental sodium for injection, United States Pharmacopeia [USP], Abbott Laboratories). Although
compliance with this instruction varies, the practice at our institution is to follow this recommendation. Bulk
solutions are prepared for convenience and frugality. Although thiopental is often used to induce anesthesia, the
daily requirement is difficult to predict, and this often results in the preparation of excess solution. The cost
advantages gained by preparing the drug in bulk versus using more expensive site-of-use prepared kits or
syringes are lost if sizable quantities of thiopental are discarded each day. Extending the shelf-life of prepared
thiopental would result in a significant savings of both time and money.
Data to support this apparently arbitrary guideline are scarce. Empiric observations from the 1940s, soon after
thiopental's introduction into clinical practice, suggest that solutions of thiopental retain their potency for periods
ranging from several days to several weeks [1-3]. Years of experience and the results of several reports [4-8] also
support these observations and seem to justify the use of thiopental beyond the recommended limit; however, the
detection methods used in these studies have limitations.
In addition to chemical stability, sterility is also an important consideration in the storage of prepared solutions.
Guidelines in USP XXIII recommend unpreserved parenteral preparations be used within 24 hours unless data
are available to support longer storage [9]. Although no commercial thiopental kit contains a preservative, the
alkaline pH of thiopental (>11) has bactericidal properties [10] that deter colonization by microorganisms. Several
studies on the growth of organisms in thiopental have been performed [1-4,8,11,12]. Recent investigations have used
either trypticase soy agar [11,12] or blood agar [8] to culture contaminant organisms in inoculated thiopental

solutions. Both of these media support the growth of Gram-positive and Gram-negative bacteria, cocci and bacilli,
and slow-growing fungi (e.g., Candida albicans). However, no study has evaluated the stability and sterility of
thiopental solutions prepared in batch solutions that are subsequently drawn into unit-dose syringes under sterile
conditions for storage and ultimate clinical use.
As pharmacy costs come under increasing scrutiny, minimizing costs has become an important aspect of an
anesthesiologist's practice. Bulk preparation of thiopental by pharmacists from 5-g kits and subsequent sterile
dispensing to 20-mL syringes could prove to be a cost-effective and more convenient alternative to site-of-use
preparations (i.e., unit dose 500-mg kits or ready-to-mix prefilled syringes) if they can be proven to be both stable
and sterile.
The purpose of this study was to provide data to support the practice of storing and using solutions of thiopental
in pharmacy-prepared syringes beyond the recommended 24-hour time limit. The study had two objectives: to
examine both the chemical stability and sterility of solutions of thiopental stored in syringes. Samples were stored
at both room temperature and under refrigeration and were analyzed over a seven-day period. We investigated
the stability by measuring absolute concentrations of both thiopental and pentobarbital using high-performance
liquid chromatography (HPLC). Pentobarbital is the primary metabolite of thiopental in vivo and therefore a
potential product of its degradation in solution. These same samples were also cultured in the hospital
microbiological laboratory for microbial growth.
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Methods
Standard solutions of sodium pentobarbital (50 micro g/mL) and the internal standard 5-ethyl-5-p-tolylbarbituric
acid (Aldrich, Milwaukee, WI), were prepared in HPLC-grade methanol (B & J Brand, Baxter, Santa Ana, CA).
Internal standard and pentobarbital solutions were stored at -20[degree sign]C between uses. Solutions of
thiopental used to construct a daily calibration curve using defined concentrations were prepared by
reconstituting a 5-g Pentothal[registered sign] kit with 100 mL sterile water for injection (SWFI) (final
concentration 5%, 50 mg/mL). Diluting this stock solution with SWFI, five different concentrations were prepared:
35 mg/mL, 30 mg/mL, 25 mg/mL, 20 mg/mL, and 15 mg/mL. Eight vials of each concentration of the thiopental
standards were made on Day 0. One set of these standards was set aside for immediate analysis to construct
the calibration curve for the Day 0 analysis; the remaining sets were frozen at -20[degree sign]C to retard
decomposition, and a single set was thawed and used on each subsequent day to construct that day's calibration
curve for thiopental. Internal standard was pipetted into every plastic conical centrifuge tube used in this study
and dried under a stream of nitrogen gas. On each day, a set of the thiopental standard solutions (35 mg/mL, 30
mg/mL, 25 mg/mL, 20 mg/mL, and 15 mg/mL) was thawed, and a fixed quantity of each concentration was
pipetted into an individual conical tube (with dried internal standard). Pentobarbital in methanol (50 micro g/mL)
was added daily to five additional tubes (containing internal standard) to make pentobarbital standards with
concentrations of 50 micro g/mL, 25 micro g/mL, 12.5 micro g/mL, 5 micro g/mL and 2.5 micro g/mL. The mobile
phase was prepared by combining methanol and 0.01 M potassium phosphate (1:1) at a pH of 4.4 +/- 0.1.
In accordance with manufacturer's instructions, thiopental (Pentothal[registered sign], USP 5 g kit, Abbott) was
reconstituted under a laminar flow hood with 200 mL of SWFI using standard sterile technique (final
concentration 2.5%, 25 mg/mL). Twenty-milliliter aliquots of the solution were drawn into sterile 30-mL Luer lock
syringes, capped, and labeled in the same manner and by the same pharmacist who routinely prepares
thiopental for the department. In this fashion, 120 study syringes were prepared on Day 0. Eight syringes (Day 0)
were set aside for immediate HPLC analysis, the remaining 112 study syringes were randomly but equally
divided into two groups and stored at either room temperature (22 +/- 1[degree sign]C) [13] or under refrigeration
(3 +/- 1[degree sign]C). These specimens were marked Day 1 to Day 7, corresponding to the number of days
until analysis.
On each day of the study, 10 mL of the thiopental solution was aseptically removed from each of the study
syringes. The pH of all samples was measured. Standard solutions prepared as described above and the study
solutions from the marked syringes were measured using a modification of the HPLC method described by
Houdret et al. [14] Analysis was performed on a reverse-phase micro Bondapak C18 HPLC column (3.9 x 300 mm;
Waters Assoc., Millipore, MA) that was equilibrated with mobile phase at a flow rate of 1.5 mL/min. Ultraviolet
absorbance was monitored at 212 nm using a variable wavelength ultraviolet detector (Waters Assoc.).
Thiopental and pentobarbital concentrations were determined by comparing the peak area ratios (i.e., the ratio of
the area under the thiopental or pentobarbital peak to that of the area under the internal standard peak) of the
study syringe solutions to a calibration curve obtained concurrently from peak area ratios of known standard
concentrations of thiopental and pentobarbital.
After removing a 10-mL aliquot of thiopental from each syringe for HPLC and pH analysis, the remaining 10 mL
of each sample was sent to the medical microbiological laboratory for culture using the same method used daily
to culture and identify microbial growth in routine samples of fluids, tissues, and devices (e.g., catheter tips).
Thiopental (0.3 mL) was inoculated into a thioglycolate medium and incubated at 35[degree sign]C in air. Each
inoculate was examined for growth every 24 h for 5 days. At the end of 5 days, a sample from the thioglycolate
medium was swabbed onto chocolate agar plates and incubated at 35[degree sign]C with 5% CO2. This
microbiological method supports the growth of Gram-negative and Gram-positive bacteria and fungi. Each plate

was examined every 24 h for an additional 3 days. Growing organisms were identified using a computerized
analyzer (VITEK; BioMerieux, Hazelwood, MO).
The total number of thiopental syringes that were prepared but not used in our surgical suite over an 8-mo period
was recorded. Outdated syringes, including unused syringes and those returned unused from anesthesia carts,
were collected from the operating room pharmacy refrigerator. Cost per syringe was calculated from material and
labor costs. The total annual expense of discarded thiopental was extrapolated from the product of the number of
unused syringes and cost per syringe.
Data obtained from this study includes thiopental and pentobarbital concentrations, pH values, and presence or
absence of microbial growth from each thiopental syringe examined from Day 0 to Day 7. Longitudinal
concentration data (mean +/- SEM) were analyzed by using repeated-measures analysis of variance (ANOVA) to
detect overall statistical differences in concentration over time. When statistical significance was detected, post
hoc multiple comparison analysis was performed using Bonferroni's correction [15] to determine on what day the
concentration or ratio changed statistically. The a priori level of significance was established at P <or=to 0.05.
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Results
The HPLC-derived concentrations for thiopental and pentobarbital are expressed as a pooled mean (+/- SEM) for
each day and each temperature group. There was a decrease in thiopental concentration in the room
temperature samples over time compared with the refrigerated samples (P <or=to 0.05, repeated measures
ANOVA) (Figure 1). Although there were no specific differences in thiopental concentration between the
refrigerated and room temperature syringes detected on any day using a Bonferroni post hoc test (most likely the
result of the variability in the analytic method), the significant trend toward a decrease in room temperature
thiopental concentration over time using repeated-measures ANOVA is a far more important finding. There were,
however, significant intragroup differences within the room temperature group. Specifically, thiopental
concentrations for Day 1 differed from those for Day 2, and Day 1 differed from Day 7 (P <or=to 0.05). The
absence of similar differences (for example, between Day 0 and Day 7) is probably the result of the large
standard deviation among daily means, representing HPLC chatter (i.e., variations for technical reasons,
generally pipetting variance).

Figure 1
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The pH values of both the room temperature and refrigerated thiopental samples did not change significantly
over the study (Table 1).

Table 1
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Sodium pentobarbital was present in all thiopental solutions analyzed in this study. Pentobarbital concentrations
did not change significantly in either the room temperature or refrigerated solutions during the 7-day study
(Figure 2).

Figure 2

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In the room temperature samples, a peak with a retention time of 3.3 min increased linearly in height over the 7
days of the study (Figure 3). This peak represents a compound with physicochemical properties different from
either thiopental or pentobarbital. The method we used did not allow for identification of this substance or to
determine its chemical activity. The timing of its appearance is consistent with the degradation of thiopental. No
absorption peak was noted at this retention time in the refrigerated samples.

Figure 3
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The thiopental concentration of the room temperature samples decreased below the USP-accepted quantity of
drug (i.e., 93% of the labeled concentration, 23.25 mg/mL) on Day 6. The refrigerated samples remained above
this level throughout the study period. Using linear regression analysis, it was extrapolated that the thiopental
concentration of the refrigerated samples would decreased below this level after 22 days.

There was no bacterial growth in thiopental samples until Day 6 and Day 7, when the growth of two
staphylococcus species was detected (Table 2). All of the thiopental solutions were clear and precipitate-free.

Table 2
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For an 8-mo period (October 1994-May 1995), outdated syringes (older than 24 h) were collected and counted. A
monthly average of 263 +/- 38 syringes was prepared but not used. This is undoubtedly a conservative Figure
becauseit is known that many unused syringes were thrown away without being included in this average. The
per-syringe cost of thiopental, supplies, and labor are shown in Table 3. These costs were extrapolated to an
annual waste of $6880. For the purpose of comparison, the list costs of thiopental in 500-mg kits and ready-tomix prefilled syringes designed for site-of-use preparation are included in this table. Preparing syringes in-house
from 5-g kits represents a significant savings over either of these unit dosage forms. The actual cost paid by
hospitals or clinics for these preparations may vary because of contractual agreements with the manufacturer or
distributor.

Table 3

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Discussion
The USP describes the stability of a therapeutic drug as the extent to which a product maintains its original
properties during a specified time period. The five factors usually considered when evaluating the stability of a
drug are chemical, microbiological, physical, therapeutic, and toxicological stability [9]. This study focuses on the
chemical and microbiological aspects of the stability of thiopental sodium.
To meet the criteria for chemical stability, a drug must remain within specified limits of chemical integrity and
labeled potency. Standard solutions of thiopental, as defined by USP XXIII, should contain no less than 93.0%
(23.25 mg/mL) of its labeled concentration (25 mg/mL) [16]. Because solutions of barbiturates degrade relatively
rapidly over time [17], thiopental sodium is best stored as a crystalline powder [18]. Numerous studies and years of
clinical experience, however, have suggested that solutions of thiopental retain their potency for periods of up to
three months, depending on method of preparation and storage [1-8].
Robinson [5] found that the appearance of precipitate in thiopental correlated with a change in melting point. He
admitted, however, that melting point studies cannot discern the nature of the precipitates. In 1961, Jones et
al. [6] conducted a stability study using spectrophotometry. After 10 days, they reported a 93% potency for room
temperature solutions and 100% for the refrigerated samples [6]. Also using spectrophotometric analysis, Das
Gupta et al. [7]confirmed the stability of thiopental stored in plastic syringes. Neither melting point determination
studies nor spectrophotometric analysis are typically indicative of chemical stability [19] because they cannot
differentiate between the parent compound and degradation products with similar melting points or absorbance
characteristics. We used HPLC to more accurately measure the degradation of thiopental and the accumulation
of pentobarbital. HPLC is a more suitable test because it can separate solutions based on physicochemical
characteristics of the components [20].
In this study, we found a significant trend toward a decline in thiopental concentration over a seven-day period
(especially the room temperature samples). Using the slope derived by linear regression (see Figure 1), room
temperature samples of thiopental are predicted to fall below the USP 93.0% minimum on Day 6. Similar linear
regression predicts that the refrigerated samples would remain above the USP recommended 93.0% minimum
for 22 days.
Even as the thiopental deteriorated, particularly within the room temperature samples, there was no statistically
significant increase in the concentration of pentobarbital in either group of samples (Figure 2). We therefore
concluded that spontaneous degradation of thiopental does not occur primarily through its desulfonation to
pentobarbital, as occurs metabolically. The three positive cultures obtained on the last two days of this study do
not seem to represent growth within thiopental; it is more likely the result of laboratory contamination. True
colonization should have occurred earlier rather later in the study. A study by Highsmith et al. [11] shows that S.
epidermidis and S. hemolyticus are intolerant of the alkaline pH of thiopental and do not survive for 24 hours. In
fact, of the organisms Highsmith et al. studied, the only medically important pathogens that tolerate the alkaline
environment of thiopental were enterococcus and Candida albicans, and these survived only for 24 hours [11].
From these findings, we conclude that thiopental reconstituted and transferred to syringes under a sterile hood
using USP guidelines remains sterile for up to seven days when stored either in a refrigerator or at room
temperature.
Strict adherence to the manufacturer's recommendations that unused thiopental solutions be disposed after 24
hours can represent a significant monetary waste for hospitals that use the 5-g bulk preparation of thiopental.

This study demonstrates that this wastage is unnecessary. In addition, batches of syringes prepared from bulk
solutions can be placed in many of the pharmacy "vending" machines or other narcotic-accounting devices as
per regulatory requirements and thereby reduce the pharmacy costs for the more expensive site-of-use prepared
unit doses of thiopental (i.e., 500-mg kits and ready-to-mix prefilled syringes). By facilitating the use of thiopental
in a more economical and convenient fashion, it is possible to add a less costly and equally accessible option to
the more expensive induction drugs.
In summary, syringes of thiopental prepared for clinical use by an operating room pharmacy under a sterile hood
remain sterile and chemically stable for six days at room temperature and much longer if refrigerated. These
findings could lead to significant monetary savings over time if bulk solutions are prepared by the pharmacy and
used until depleted. They also mean that prepared syringes can be left in strategic locations at room temperature,
ready for use in emergency situations (e.g., in obstetric delivery rooms, trauma rooms, etc.), for up to six days
with reliable potency.
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