Chemical Composition of Everyday Products
Chemical Composition of Everyday Products
John Toedt, Darrel Koza, Kathleen Van Cleef-Toedt
Greenwood Press
88 Post Road West, Westport, CT 06881
0313325790 $49.95 www.greenwood.com
Both high school and college collections strong in chemistry will want to add this helpful analysis to their collection: over 100 groups of products receive attention to not only chemical composition, but historic product use, functions, and related issues. Ten lab exercises provide students with observation keys, making CHEMICAL COMPOSITION OF EVERYDAY PRODUCTS the perfect supplemental text for introductory chemistry courses. Easy to access and understand, relating to everyday materials from soap and cosmetics to baby products..what more could one wish for?
Chemical Weapons Convention chemicals analysis; sample collection, preparation, and analytical methods
Chemical Weapons Convention chemicals analysis; sample collection, preparation, and analytical methods.
Ed. by Markku Mesilaakso.
John Wiley & Sons
2005
462 pages
$335.00
Hardcover
UA12
Contributors from the Organization for the Prohibition of Chemical Weapons, based at The Hague, and from other organizations and universities, explain how to verify international compliance with the Chemical Weapons Convention in principle, using analytical chemistry and related strategies and methods. The Organization has designated 18 analytical laboratories around the world as capable of analyzing samples of chemicals used in chemical weapons, and most of the chapters here describe the analytical methods used in them to identify target chemicals from environmental and human origin samples. They also discuss the procedures and strategies for on-site sampling, for example during the inspection of facilities, and either analyzing substances on-site or sending them to other sites for analysis.
([c] 2005 Book News, Inc., Portland, OR)
Chemical Composition of the Essential Oil from Rhizomes of Rhodiola rosea L. Grown in Finland
Abstract
The essential oil of Rhodiola rosea L., from rhizomes cultivated in Finland was analyzed by GC and GC/MS methods in Hungary. The air-dried rhizomes contained 0.04% essential oil. Thirteen components which were characterized in the oil were mainly monoterpenoid (84.3%). Myrtenol (36.9%), trans-pinocarveol (16.1%), geraniol (12.7%) and dihydrocumin alcohol (12.1%) were the most abundant volatiles detected in the oil. Myrtenol, geraniol and linalool were identified as the most important rose-like odor compounds which is important to give a pleasant rose-like scent to these Nordic rhizomes.
Key Word Index
Rhodiola rosea, Crassulaceae, rose root, essential oil composition, octanol, trans-pinocarveol, myrtenol, geraniol, cumin alcohol.
Plant Name
Rhodiola rosea L., (Arctic Root, Rose Root), Crassulaceae family
Source
Seeds of Rhodiola rosea vas collected more than 10 years ago at the border of Norway and north Finland by the staff of Särkä Nursery (922110 Raahe, Finland). The marketed seedlings were transplanted into the observation plots of Agrifood Research Finland, Mikkeli during 1994. Seedlings from own plants were transplanted into experimental fields in spring 1997.
Plant Part
Rhodiola rosea L. plants have been cultivated at the Agrifood Research Centre in Mikkeli (grid reference: 61° 44′ N, 27° 18′ E) during 1997-2002 in Finland. It is a perennial plant, reaching a height of 12 to 30 in (max. 70 cm) in cultivation and its full flowering time is in June, having yellow blossoms. The slow growing plants need five years to reach suitable root yields. The plants have a thick rhizome with rose-like fragrance when cut. About 30% of the total fresh weight of the rhizome consists of thinner and hairy root, 15-30 cm in length.
Previous Work
Rhodiola rosea is widely distributed in Arctic and circumpolar areas in high altitudes in mountainous regions throughout Europe and Asia. It is a popular plant in traditional medical systems with a reputation for stimulating the nervous system (1), decreasing depression (2) and preventing high altitude sickness (3). Its Russian name is “golden root,” and the plant has been studied intensively in Russia and Scandinavia for more than 35 years (4,5). Its claimed benefits include antidepressant, anticancer, cardioprotective and central nervous system enhancement. The roots have different chemical compounds, of which its pharmacological effects supposed are belonging to phenylpropanoids, like rosavin, rosin, rosarin, and to phenylethanol derivates, like salidroside (6,7).
Roots of R. rosea have been analyzed mainly for the above mentioned compounds (8-10), however little is available concerning the rose-like fragrant compounds of the roots. Recently, terpenes and aroma volatiles have been isolated from the rhizomes of R. rosea of Norwegian origin (11). The dried rhizomes were found to contain 0.05% essential oil with decanol (30.38%), geraniol (12.49%) and p-mentha-1,4-dien-7-ol (5.10%). Geraniol was identified as the most important rose-like odor compound.
Present Work
Whole roots (rhizomes and hairy roots) were dug up on September 21, 2001. The plants were washed, sliced and dried in an air-forced commercial drier at 40°C temperature. The dried rhizomes were ground and water distilled, using a modified Clevenger-type apparatus to produce the oil (in 0.04% yield) at the Technical University Budapest in Hungary.
For identification of the oil components, analytical gas chromatography was performed a Shimadzu GC-14B capillary gas chromatograph apparatus with FID detector and Supelco SE 30 quarz column (25 m x 0.25 mm, 0.25 µm film thickness) at the Szent István University, Department of Medicinal and Aromatic Plants. Temperature was programmed from 110°-220°C at 8°C/min. Nitrogen was used as carrier gas at a flow rate of 1 mL/min. Injector temperature was 220°C and detector was 250°C.
GC/MS analyses were carried out on a Finnigan Mat GCQ with RESTEC-5 column (30 m x 0.25 mm, 0.32 µm film thickness) at the Naturland Hungary Ltd. Electron impact MS, and the ionization energy was 70 eV, carrier gas was He 75:1 splitter condition. GC/MS analyses were carried out on a Hewlett-Packard 5890/II GC-5971A MSD with Supelcowax 10 column, 60 m x 0.25 mm, 0.32 µm film thickness as well. Ionization energy was 70 eV at the Szent István University, Department of Food Chemistry.
Tempe rature programming was from 60°-240°C at 4°C/min. Component identifications were made by comparison of their mass spectra and retention indices with those of authentic compounds, and with data in the NIST and NISTPlus Library as described by Héthelyi et al. (12).
As show in Table I, about 99.7% of the oil was identified. The oil was characterized by large amount (84.3%) of monoterpenoid components with myrtenol (36.9%), trans-pinocarveol (16.1%), geraniol (12.7%), cumin alcohol (12.2), linalool (2.7%), dihydrocumin alcohol (2.1%) and perilla alcohol (1.7%). We have identified some other interesting compounds (14.9%) such as: octanol (13.6%) and 6,6-dimethyl-bicyclo-[3,1,1]-hept-2-ene-2-carboxaldehyde (1.0%).
References
1. A.S. Saratikov, Central nervous system stimulants of plant origin. Stimulyatory Tsent. Nerv. Sist. Edits., A.S. Saratikov, pp 3-23, Izd. Tomsk. Univ., Tomsk, USSR (1966).
2. M. Furmanowa, B. Kedzia, M. Hartwich, J. Kozlowski, A. Krajewska-Patan, A. Mscisz and J. Jankowiak, Phytochemical and pharmacological properties of Rhodiola rosea L. Herba Polon., 45, 108-113 (1999).
3. E.P. Shirokov, D. Badgaa and I.V. Kobozev, Essential oil content in plants used in the production of tonics. Izvestiya Timiryazevskoi Selskokhozyaistvennoi Akademii, 3, 187-91 (1980).
4. Z. Ramazanovand M.M.B. Suarez, New Secrets of Effective Natural Stress and Weight Management Using Rhodiola rosea and Rhododendron caucasicum. ATM/Safe Goods Publishing, East Canaan, Connecticut (1999).
5. C. Germane and Z. Romazanov, Arctic Root (Rhodiola rosea) The Powerful New Ginseng Alternative. Kensington Books, Kensington Publishing Corp. (1999).
6. P.P. Brown, P.L. Gerbarg and Z. Ramazanov, Rhodiola rosea A Phytomedicinal overview. HerbalGram, 56, 40-52 (2002).
7. G.S. Kelly, Rhodiola rosea: a possible plant adaptogen. Altern. Med. Rev., 6, 293-302 (2001).
8. A.A. Spasov, G.K. Wikman, V.B. Mandrikov, I.A. Mironova and V.V. Neumoin, A double-blind, placebo-controlled pilot study of the stimulating and adaptogenic effect of Rhodiola rosea SHR-5 extract on the fatigue of students caused by stress during an examination period with a repeated low-dose regimen. Phytomedicine, 7, 85-89 (2000).
9. V.A. Kurkin and G.G. Zapesochnaya, Chemical composition and pharmacological properties of Rhodiola rosea. Chem. Pharm. J. (Moscow), 20, 1232-1244 (1986).
10. A.G. Dubichev, B.A. Kurkin, G.G. Zapesochnaya and E.D. Vornotzov, Study of Rhodiola rosea root chemical composition using HPLC. Cemico-Pharmaceutical J., 2, 188-93 (1991).
11. J. Rohloff, Volatiles from rhizomes of Rhodiola rosea L. Phytochemistry, 59, 655-61 (2002).
12. É.B. Héthelyi, K. Korány, K. Jokela, B. Galambosi, J. Domokos and J. Pálinkás, Determination of the Essential Oil Composition and the Bitter-Value of Acorus calamus by GC, GC/MS Methods and Sensoric Evaluation. Medicinal Plant Research and Utilisation 2002.10th National Conference on Medicinal Plants. KECSKEMET 2002. November 13-15. Book of Abstracts, p. 173 (2002).
Éva B. Héthelyi*
Szent István University, Department of Medicinal and Aromatic Plants, and Naturland Hungary Ltd. Budapest, Hungary
Kernél Korány
Szent István University, Department of Food Chemistry, Budapest, Hungary
Bertalan Galambosi
Agrifood Research Finland, Environmental Research, Kanla, Mikkeli, Finland
János Domokos and János Pálinkás
BME Technical University Budapest, Department of Physical Chemistry, KHV Ltd. Budapest, Hungary
* Address for correspondence
Received: February 2003
Revised: May 2003
Accepted: August 2003
1041-2905/05/0006-0628$6.00/0-© 2005 Allured Publishing Corp.
Copyright Allured Publishing Corporation Nov/Dec 2005
Provided by ProQuest Information and Learning Company. All rights Reserved
Xylanase Lowers Chemical Load, Boosts Brightness of Kraft Pulp
A Canadian softwood kraft pulp mill showed good results helping to increase brightness, reduce chemical charge and lower the effluent load
Due to environmental pressures and market forces demanding low adsorbable organic halide (AOX) pulp, a softwood kraft pulp mill is investigating options to reduce chlorine and chlorine dioxide consumption in its bleach plant.
One option now being used on a semi-permanent basis is the use of the xylanase enzyme as a pre-bleaching chemical. Work has shown that enzymes can be used in the treatment of unbleached pulp for delignification and brightening.1
The mill began to apply enzymes in August 1992 following completion of the bench scale and mill scale trials in 1991.2 The bench scale trials done showed an average of 12-15% reduction in total equivalent chlorine for the (C/D30)(Eo)(D)(E)(D) bleaching sequence while the mill scale trial showed an average reduction of 15-23%. Mill scale trials showed that pulp quality as measured by tear, tensile and burst strength was not affected.
While the initial mill scale trial showed similar or better improvement, most of the work was done prior to hydrogen peroxide application in the first extraction stage. Work using enzymes in conjunction with hydrogen peroxide showed much improved pulp brightness.3 It has also been shown that peroxide and enzymes in combination can be used to lower kappa factor for improved pulp brightness, effluent quality and viscosity.4
The objective of this work is to further substantiate that enzymes are effective in reducing the total equivalent chlorine with 0.4% hydrogen peroxide application in the first extraction stage. At reduced equivalent chlorine, the discharge of AOX in treated effluent should also decrease. Depending on how much the total equivalent chlorine decreases, bleaching costs may decrease and the option of increasing production without expanding chlorine dioxide generator capacity becomes viable.
Many Parameters Impact Enzyme Effectiveness
Enzyme use in Canadian mills was initiated in 1991 mainly to enhance the bleaching process.5 Since then some mills have been using enzymes consistently with varying objectives: reducing the chlorine consumption to lower AOX or BOD; or; reducing the bleaching cost.
Enzymes are protein catalysts found in living organisms. These catalysts tend to be highly specific in terms of the reaction they catalyze and the physical conditions under which they operate effectively.6 The idea of enzyme bleaching first emerged in 1984.7 Later, it was reported that enzymes could enhance pulp bleaching and save chemicals.8-10 Due to its specificity, xylanase is used to bleach pulp. Other enzymes are also available, but xylanase was used in this trial.
Xylanase enzymes are produced from microorganisms. These enzymes hydrolyze the xylan present in the brownstock fiber, rendering the lignin more accessible to the bleaching chemicals as well as allowing the bleached lignin to diffuse out of the fiber more readily. This results in higher brightness pulp for a given charge of chemicals or reduced chemical usage for constant brightness target.
Due to the specific nature of the enzyme, many parameters can affect its effectiveness. The most important ones are application rate, reaction temperature, pH, treatment time and adequate dispersion of enzyme.
For the mill where the trials were done, the optimum and safe operating conditions for applying the enzyme were determined by the supplier (Table 1).
The pH of the brownstock is dependent on the washing efficiency of the brownstock washer. The initial pH in the brown screen accepts line typically varies between 9.5-10.5. To control the pH in the specified range, very dilute sulfuric acid solution is added on the discharge of the unbleached decker sheet and controlled to maintain the target at 6.2.
Based on work conducted by the supplier, a minimum of two hours of residence time is required to gain maximum enzyme benefit. To attain two hours, the screened high-density level is maintained at 40%+. Although there are times when the level goes below the specified set point, it should be noted that 75-80% of the enzyme benefits are achieved in the first 30 min.
The supplier specifies the application rate, based on its knowledge of the concentration required for effective treatment.
Temperature is maintained by adjusting the volume of cold water used in the unbleached decker (UBD). The fact that the optimum temperature for effective enzyme treatment was similar to the chlorination stage operating temperature greatly reduced the complexity of adjusting the temperature for treatment.
Although mixing and channeling has not been specified in the parameters being monitored, both are important in ensuring effective treatment. Mixing is important to ensure even distribution and treatment of enzyme and pulp. Channeling of stock through the screened high-density storage is also critical, Unpublished work done by the supplier has shown that stock can channel through the storage tank and reduce the residence time, thus decreasing the enzyme treatment time and the optimum benefit achievable.
Kraft Pulping Process and Equipment
Figure 1 shows the typical UBD and screened high-density arrangement at the mill where the trial was conducted. Stock is transferred from the brownstock high-density storage tank via the brown screening system to the UBD. The stock is thickened to about 11% consistency and discharged into a chute leading to a thick stock pump. The pump transfers the stock to a screened high-density storage tank.
From there it is pumped via a screened stock chest to the bleach plant. Most of the metal parts are constructed of 316L and 317L, thus reducing the possibility of corrosion in the presence of dilute sulfuric acid.
Enzyme and sulfuric acid diluted with brown white water and fresh water are added through a shower bar onto the sheet discharging from the UBD. The enzyme addition rate is controlled by the brown screen accepts production rate. The sulfuric acid addition is adjusted through a variable speed pump receiving a signal from the pH probe located at the discharge point of the thick stock pump. A constant flow of fresh water and brown white water are applied continuously while the enzyme system is operating.
The enzyme skid is designed to provide self-contained control of the dosage of acid and enzyme that is mixed and dispersed. The chemicals are then diluted with water and fed into a shower bar on the discharge side of the brown decker, just before the doctor board.
The system is designed to allow the supplier to log and analyze data from a remote location. There are three principal modules to the equipment: field addition system; monitoring and control station; acid handling system
Field addition system: The field system is a skid (28 in. x 30 in.) with piping, pumps and electrical controls. There is an independent single loop digital controller, which uses field inputs (stock flow, pulp pH, pulp temperature, and stock tank level) to control the speed of positive displacement acid and enzyme pumps, and to provide safety and operational interlocks.
Monitoring and control station: The monitoring and control station uses an industrial PC as an interactive workstation to monitor and control the field addition system from the mill control room. It uses a touch screen and a customized user-friendly graphical program to provide process displays, alarming, trending, reporting, data logging and troubleshooting help.
Acid handling system: The use of enzymes involves adding acid to the brownstock. The supplier has provided a separate acid pumping skid for use with an acid tanker. It pumps the acid through a mill-installed acid line to the addition system. The acid pump and a safety shutoff valve are controlled and interlocked by the enzyme addition system.
Experimental Parameters
Enzymes: As noted, xylanase is used at the mill. The enzyme is supplied as a liquid with a xylanase activity of 3,600 XU/mL. The enzyme solution is stable at room temperature and contains a sodium benzoate preservative. Enzyme solution analysis is presented in Table 2.
Screened chips, predominantly hemlock/fir (Table 3), are cooked in two identical continuous single vessel Kamyr hydraulic digesters. From the digester the blown stock goes through knotters, brown stock washers and a brown screening system before enzyme addition and pH adjustment on the UBD.
Sulphuric acid: The acid is supplied to the skid as 93% W/W strength. The acid mixes with the brown white water to lower its strength below 0.5% weight-to-weight ratio prior to distributing onto the pulp sheet discharging the UBD.
Bleach Plant Operating Procedure
Bleaching: The enzyme treated pulp is bleached in a five stage bleaching sequence: D/CEOpD^sub 1^E^sub 2^D^sub 2^. Bleaching conditions are presented in Table 4.
Testing the additivity assumption: chemical mixtures and thyroid function
It is well established that many environmental contaminants can disrupt thyroid hormone (TH) homeostasis, which is vital during fetal development and for a variety of physiological processes in adults. Among known TH disruptors are polychlorinated biphenyls (PCBs), dioxins, and dibenzofurans, all members of the polyhalogenated aromatic hydrocarbon (PHAH) chemical family. Little is known, however, about how mixtures of such chemicals at typical environmental exposure levels may disrupt TH functions. Nor is it clear whether effects are additive, synergistic, or antagonistic–that is, whether there is interaction between constituent chemicals, whether their cumulative influence is more than the sum of its parts, or whether they cancel each other out. With respect to risk assessment, the U.S. Environmental Protection Agency’s default assumption is that the effects of chemicals in mixtures are additive. Now a team of researchers has tested the additivity assumption and found that it is relatively robust at exposure levels typical for humans [EHP 113:1549-1554].
Over a four-day period the team exposed young female rats to six different doses of a combination of 18 PHAHs comprising 2 dioxins, 4 dibenzofurans, and 12 PCBs. The team determined dose-response information for each constituent chemical before the mixture was tested. The concentration of each chemical in the mixture reflected typical concentrations measured in breast milk and in fish and other foods. The mixture was also formulated so that even at the highest mixture doses, the rats’ exposure to each constituent chemical was at or below the known no-observed-effect level for that chemical.
The mixture reduced the rats’ serum thyroxine ([T.sub.4]; the most common form of circulating TH) in a dose-dependent manner. At lower doses the effects were additive. At higher doses [T.sub.4] declined by as much as 50%, and the effects were mildly synergistic–about twice what was predicted by additivity–so that even in the upper range the effects as predicted by the additivity hypothesis came close to actual results.
Significantly, the study also showed that the mixture exerted an effect on [T.sub.4] even though concentrations of its constituent chemicals were at least an order of magnitude below their known effective doses. This indicates that considering individual chemicals in isolation may not predict their effects in mixtures because, even though chemicals may not be potent enough by themselves to cause effects, the cumulative effects of low doses of many chemicals may be enough to do so.
The multiple functions of TH, such as its role in fetal development and its regulation of metabolism and heart rate, make it vulnerable at many points. The team estimates that there could be as many as five distinct mechanisms by which chemicals exert antithyroid effects for which a reduction in circulating [T.sub.4] is the common end point.
Several factors temper the study results. One is that this study was a series of short-term exposures that did not encompass all the chemicals’ varied half-lives. The results therefore cannot be directly extrapolated to the effects of chronic exposures and may be subject to confounding by pharmacokinetic differences. Another is that thyroid disruption mechanisms in rats may not be identical to those in humans. The team is now working on testing how a more complex chemical mixture may interact with dietary iodine insufficiency to produce thyrotoxic effects.
Chemical Stability of Fentanyl in Polypropylene Syringes and Polyvinylchloride Bags
Abstract
Fentanyl is a potent synthetic lipophilic opiate agonist used to control pain as a single agent or in combination with local anesthetics. The chemical stability of the undiluted commercial solution (50 µg/mL) in polypropylene syringes or polyvinylchloride bags has never been reported. Undiluted fentanyl solution, 50 µg/mL, was aseptically transferred to polypropylene syringes or polyvinylchloride bags. Samples were then stored, either at 5°C and protected from light, or at 22°C and exposed to light, for 28 days. A stability-indicating high-performance liquid chromatographic method was used to monitor the fentanyl concentration of the samples. Color, clarity, and pH also were monitored. After storage for 28 days, there were no signs of chemical degradation of fentanyl packaged in either polypropylene syringes or polyvinylchloride bags at either 5°C or 22°C. All solutions remained colorless and clear over the course of the study. The pH did not change significantly after storage for 28 days. Fentanyl solutions, when packaged undiluted in polypropylene syringes or polyvinylchloride bags, are chemically stable for 28 days when stored either at 5°C and protected from light or at 22°C and exposed to light.
Introduction
Fentanyl may be used as a single agent or in combination with local anesthetics to control pain. The stability of fentanyl, when diluted with isotonic saline solution, has been studied over the range of concentrations from 12.5 µg/mL to 33.3 µg/mL for up to 30 days in polypropylene (PP) syringes and polyvinylchloride (PVC) bags.1-4 A pH of greater than 5.5 seems to cause sorption of the drug on PVC surfaces; refrigeration decreases this effect.5
The objective of this study was to determine the chemical stability of undiluted fentanyl when packaged in PP syringes or PVC bags and stored either at 5°C and protected from light, or at 22°C and exposed to light, for 28 days.
Materials and Methods
All chemicals and reagents used were United States Pharmacopeia-National Formulary (USP-NF) or high-performance liquid chromatographic (HPLC) grade. There was no further purification of the chemicals. Undiluted fentanyl solution (Lot 15058NJ, Abbott Laboratories Ltd., Montreal, Canada) was used for the study.
Equipment
An HPLC system was used to monitor the fentanyl concentration of the samples. A C^sub 18^ reverse phase column was used (Partisil, 4.6 × 250 mm, 10 pm; Phenomenex, Torrance, California). The HPLC system consisted of an isocratic solvent pump (Model LC-10AT[VP]; Shimadzu Corporation, Kyoto, Japan), an autoinjector (Model SIL-10A^sub XL^; Shimadzu Corporation), and a photodiode array detector (Model SPD M6A; Shimadzu Corporation). The pH measurements were determined with a calibrated pH meter (Model Accumet 25; Fisher Scientific Ltd., Nepean, Canada).
Chromatographic Conditions
The mobile phase was prepared by mixing methanol, acetonitrile, and ammonium acetate buffer in the ratio of 20:20:60. The ammonium acetate buffer was prepared by dissolving 6 g of ammonium acetate in 600 mL of HPLC-grade water and then adding 0.6 mL of glacial acetic acid. The pH of the mobile phase was adjusted to 6.6 ±0.1 using ammonium hydroxide 6 N.
Sample Preparation
Undiluted fentanyl (50 µg/mL) was aseptically transferred to PP syringes (which were then capped) or to PVC bags and stored either at 5°C and protected from light or at 22°C and exposed to light. Three samples were collected from each type of container on days 0, 7, 14, 23, and 28. Color and clarity changes were determined by visual inspection. The pH of each sample was determined with a calibrated pH meter. Samples were then frozen at -70°C and analyzed at a later date. On the day of analysis, samples were warmed to room temperature, 100 µL of internal standard was added to 1000 µL of sample, and the samples were assayed in duplicate.
Assay Validation
The stability-indicating nature of the HPLC method was confirmed by monitoring forcibly degraded fentanyl samples. For one sample, undiluted fentanyl solution was adjusted to a pH of approximately 1.5 with concentrated hydrochloric acid. A second solution was adjusted to a pH of approximately 12.0 with 5 N sodium hydroxide, while a third solution had 1 mL of 30% hydrogen peroxide added to it. The acidic and alkaline solutions were incubated in a 70°C water bath. All solutions were then subjected to chromatography at 0, 2, 21, 48, 96, 192, and 216 hours after preparation. Monitoring for interfering peaks was done at 230 nm. Peak purity was determined using multiple wavelength (220 and 230 nm) and ultraviolet (UV) spectral analyses (200 to 350 nm).
Intraday variation was measured by analysis of five replicate injections at 0, 16, and 25 hours. Interday variation was determined by comparing slopes, correlation coefficients (r^sup 2^), and peak ratios from a standard solution on five separate days. Method accuracy was determined from analysis of a recovery solution of known concentration on five separate days. Least squares linear regression was used to calculate the linearity of a concentration versus response curve.
Results and Discussion
In experiments validating the stability-indicating ability of the HPLC assay, the acidic conditions produced a faster eluting degradation peak, while the alkaline conditions produced a slower eluting peak. Oxidation of the fentanyl produced multiple faster eluting peaks. None of the degradation peaks interfered with the parent or internal standard peaks. All parent peaks were confirmed to remain pure by multiple wavelength and UV spectral analyses. Overlay of the UV spectra from parent peaks in the degradation samples on the fentanyl peak from a reference sample gave a correlation coefficient of 0.9928 or greater.
The correlation coefficient for the intraday analysis was 1.7%. Interday variations of the slopes, r^sup 2^, and peak ratios were 1.10, 0.01, and 0.97%, respectively. The average recovery result was 101.4 ± 1.9%. The concentration versus response curve was linear over the range of 5 to 50 µg/mL (r^sup 2^ = 0.9913).
All solutions remained clear and colorless over the course of the study. There was no significant change in pH in any of the solutions and no pH greater than 5.43 was measured during the study.
The results of the chemical analyses of the fentanyl samples are summarized in Tables 1 and 2. Concentrations of all solutions remained greater than 90% of the original concentration for 28 days, whether stored at 5°C and protected from light or at 22°C and exposed to light.
Conclusion
The chemical stability of undiluted fentanyl solution (50 µg/mL) packaged in PP syringes or PVC bags was determined to be at least 28 days when stored either at 5°C and protected from light or at 22°C and exposed to light.
References
1. Stewart JT, Warren FW, King DT et al. Stability of ondansetron hydrochloride and 12 medications in plastic syringes. Am J Health Syst Pharm 1998; 55(24): 2630-2634.
2. Wilson KM, Schneider JJ, Ravenscroft PJ. Stability of midazolam and fentanyl in infusion solutions. J Pain Symptom Manage 1998; 16(1): 52-58.
3. Allen LV Jr, Stiles ML, Tu YH. Stability of fentanyl citrate in 0.9% sodium chloride solution in portable infusion pumps. Am J Hosp Pharm 1990; 47(7): 1572-1574.
4. Bing CM. Extended Stability for Parenteral Drugs. Bethesda, MD: American Society of Health-System Pharmacists; 2001.
5. Sattler A, Jage J, Kramer I. Physico-chemical stability of infusion solutions for epidural administration containing fentanyl and bupivacaine or lidocaine. Pharmazie 1998; 53(6): 386-391.
Ronald F. Donnelly, MSc (Chem), BSc (Pharm)
The Ottawa Hospital (Civic Campus)
Ottawa, Canada
Address correspondence to Ronald F. Donnelly, MSc (Chem), BSc (Pharm), Department of Pharmaceutical Services, The Ottawa Hospital (Civic Campus), Ottawa, Canada K1Y 4E9. E-mail: rdonnelly@ottawahospital. on.ca
Copyright International Journal of Pharmaceutical Compounding Nov/Dec 2005
Provided by ProQuest Information and Learning Company. All rights Reserved
A Study of the Mediterranean Oregano Populations: Chemical Composition of Essential Oils of Origanum cordifolium Monbret et Aucher from Two Populations in Cyprus
Abstract
The seeds of two populations of oregano (O. cordifolium) harvested in Cyprus were sown at a research station in France. The resulting plants were planted in triplicate in a Fischer block design. The oils, which were produced by hydrodistillation, were analyzed by GC and GC/MS. The oils were found to be rich in α-terpineol (45.9-55.7%) of homogeneous composition irrespective of their origin or replicate.
Key Word Index
Origanum cordifolium, Lamiaceae, essential oil composition, α-terpineol, carvacrol, γ-terpinene.
Introduction
Ietswaart (1) drew up a detailed botanical classification of oregano species. The chemical compositions of several of Origanum species have been determined, e.g., Baser and Duman (2-9), Skoula et al. (10), Turnen et al. (11), Valentini et al. (12), Arnold et al. (13), Ravid and Putievsky (14), Akgiil and Bayrak (15), Fischer et al. (16), Vera and Chane-Ming (17), Pino et al. (18), Melegari et al. (19), Chalchat and Pasquier (20).
Origanum cordifolium is a very rare oregano found in a few parts of a mountainous massif in the west of Cyprus. It grows on stony soils in ravines, gorges and pinewood understories at altitudes between 500 m and 1500 m. In the wild it can reach a very large size but grows very slowly in the nursery. It is a prostrate creeper and is fragile, being prone to rot. In temperate climates it is sensitive to even mild winter conditions. It is weakly scented and finds use as an ornamental pot plant appreciated for its long drooping leafy stems ending in very lone inflorescences with large colored bracts.
Origanum cordifolium Monbret et Aucher was classified by letswaart (1) in the Amaracus section of Group A. The chemical composition of its oil was studied by Valentini et al. (12), who found α-terpineol, γ-terpinene and p-cymene as the main constituents. Other species belonging to the same sections and group have been studied: O. calcaratum L. and O. dictamnus L. by Skoula et al. (10), O. silymicum Davis and O. saccatum Davis by Turnen (11), and O. boissieri letswaart by Baser and Duman (2). In the oils of these species, p-cymene was present in large amounts (20-45%).
Here we report on the chemical composition of oils of O. cordifolium of Cypriot plant origin.
Experimental
Plant material: The seeds were harvested in the west of Cyprus near Roudia and Panagia in 1998. They were sown at the CNPPMAI botanical facility at Milly-la-Foret in March 1999, in three replicates in a Fischer block design. The plants were harvested in full flower, dried in the dark at ambient temperature in a ventilated room. A specimen was conserved in the CNPPMAI Herbarium.
Oil isolation: The isolation of oils was carried out at the CNPPMAI facility by use of a Clevenger-type hydrodistillation system for 3 h. The amount of material processed per distillation corresponded to a single plant when in sufficient quantity: if not, then the whole plant material of all the repeats or all the population was pooled.
GC: The gas phase chromatography analysis was carried out on a Delsi DI 200 instrument equipped with a flame ionization detector and a DBS column (25 m x 0.25 mm, df: 0.25 µm) with a split flow rate of 60 mL/min, nitrogen as carrier gas and temperature programming (5 min at 50°C and 3°C/min up to 220°C), injector temperature 220°C and detector temperature 235°C. Quantitative data were obtained from FID areapercents without the use of correction factors.
GC/MS: The oils were analyzed on a Hewlett-Packard gas Chromatograph model 6890 coupled to a Hewlett-Packard MS model 5873 equipped with an HP5 column (30 m x 0.25 mm, df: 0.25 µm) programmed from 50°C (5 min) to 300°C at 5°C/min, and 5 min hold. The carrier gas was helium (1 mL/min), injection in split mode (1/10); injector and detector temperatures 250°C and 280°C, respectively. The MS ran in electron impact mode at 70 eV, electron multiplier 2200 V, ion source temperature 230°C. Mass spectral data were acquired in the scan mode in the m/z range 33-450.
Identification was carried out by calculating retention indices and comparing mass spectra with those in data banks; personal, Adams (21), Mc Lafferty and Stauffer (22).
Results and Discussion
The yields of oils ranged from 2.1-3.4% of dry plant matter; their density and refractive index were approximately 0.950 g/mL and 1.486 g/mL, respectively. The analytical results are given in Table I. Seventy constituents were identified: 32 were quantified (greater than or equal to 0.1%), while the others were found only in trace amounts.
The oils produced from plants from the two locations, and corresponding to five replicates, had similar chemical compositions that showed the plants to be α-terpineol-type with α-terpineol content in the range 45.9-55.6%. Three other major constituents were carvacrol, γ-terpinene and p-cymene (6.2-13.8%, 6.0-9.0% and 3.2-5.8%, respectively). All the other constituents were present in very small amounts. The most abundant included monoterpenes, α-pinene (1.8-3.1%), camphene (2.6-4.2%), sabinene (1.5-2.1%), myrcene (1.2-2.0%) and limonene (1.5-2.1%), and monoterpenols, borneol (5.1-8.2%) and terpinen-4-ol (0.7-3.4%). The only quantifiable sesquiterpenes were β-caryophyllene and its oxide, and germacrene D. The proportions of the two most abundant components showed the oils to be of fairly uniform composition, with a slight predominance of carvacrol in batch 98-007-010 and of γ-terpinene in batch 98-006-007. Generally, α-terpineol was present in small amounts and in few species, e.g., O. majorana (0.6%) (6) and O. vulgare (0.1%) (19) and at slightly higher levels in O. majorana ssp. tennuifolium (7.1%) (23) and O. vulgare (9.3%) (24). However, it can be found in much larger amounts in O. vulgare and O. ramonense at 68% (19) and 42% (25), respectively. The combined presence of carvacrol, γ-terpinene and p-cymene is not surprising as they are linked by their biosynthetic pathways as studied by Croteau in Thymus vulgaris (26) and observed in O. gratissimum by Yayi (27).
This study reveals a marked uniformity in chemical composition in the two populations of O.cordifolium found in Cyprus. They are both rich in α-terpineol, but this feature is not specific as it is shared by O. vulgare and O. ramonense.
References
1. J.H.A. letswaart, Taxonomic revision of the genus Origanum (Labiatae). Leiden Botanical Series, Leiden University Press,The Hague, Netherlands, 4(1980).
2. K.H.C. Baserand H. Duman, Composition of the essential oils of Origanum boissieri letswaart and Origanum bargyli Mouterde. J. Essent. Oil Res., 10, 71-72(1998).
3. K.H.C. Baser, N. Ermin, M. Kürkçüoglu and G. Tümen, Essential oil of Origanum hypericifolium O. Schwarz & RH. Daws. J. Essent. Oil Res., 6, 631-633 (1994).
4. K.H.C. Baser, G.Tumenand H. Duman, Essential oil of Origanum acutidens (Hand.-Mazz.) letswaart. J. Essent. Oil Res., 9, 91-92 (1997).
5. K.H.C. Baser, N. Ermin, T. Özek, B. Demircakmak, G. Tümen and H. Duman, Essential Oil of Thymbra sintenisii Bornm. & Aznav. Subsp. isaurica RH. Davis and Origanum leptocladum Boiss. J. Essent. Oil Res., 8, 675-676 (1996).
6. K.H.C. Baser, N. Kirimer and G. Tümen, Composition of the essential oil of Origanum majorana L from Turkey. J. Essent. Oil Res., 5, 577-579 (1993).
7. K.H.C. Baser, G. Tümen, T. Özek and E. Sezik, Composition of the essential oils of Turkish Origanum species with commercial importance. J. Essent. Oil Res., 5, 619-623 (1993).
8. K.H.C. Baser, T. Özek, M. Kürkçüoglu and G. Tümen, Essential oil of Origanum micranthum Vogel. J. Essent. Oil Res., 8, 203-204 (1996).
9. K.H.C. Baser, T. Özek, M. Kürkçüoglu and G. Tümen, Essential oil of Origanum vulgare ssp hirtum of Turkish origin. J. Essent. Oil Res., 6, 31-36 (1996).
10. M. Skoula, P. Gotsiou, G. Naxakis and C.B. Johnson, A chemosystematic investigation on the mono and sesquiterpenoïds in the genus Origanum (Labiatae). Phytochemistry, 52, 649-657 (1999).
11. G. Tümen, K.H.C. Baser and T. Özek, Essential oil of Origanum saccatum PH. Daws. J. Essent. Oil Res., 7, 175-176 (1995).
12. G. Valentini, N. Arnold, B. Bellomaria and H. J. Arnold, Study of the anatomy and the essential oil of Origanum cordifolium, an endemic of Cyprus. J. Ethnopharm., 35, 115-122 (1991).
13. N. Arnold, B. Bellomaria and G. Valentini, Composition of the essential oil of three different species of Origanum in the Eastern Mediterranean. J. Essent. Oil Res., 12, 192-196 (2000).
14. U. Ravid and E. Putievsky, Essential oils of Majorana syriaca, Coridothymus and Satireja thymbra plants growing wild in Israel. Planta Med., 49, 248-249 (1983).
15 A. Akgül and A. Bayrak, Constituents of essential oils from Origanum species growing wild in Turkey. Planta Med., 53, 4, 114 (1987).
16 N. Fischer, S. Nitz and F. Drawert, Original flavour compounds and the essential oil composition of marjoram (Marojana hortensis Moench). Flav. Fragr. J., 2, 55-61 (1987).
pH Dependence of Amide Chemical Shifts in Natively Disordered Polypeptides Detects Medium-Range Interactions with Ionizable Residues
ABSTRACT
A growing number of natively disordered proteins undergo a folding/binding process that is essential for their biological function. An interesting question is whether these proteins have incompletely solvated regions that drive the folding/ binding process. Although the presence of predominantly hydrophobic buried regions can be easily ascertained by high-sensitivity differential scanning calorimetry analysis, the identification of those residues implicated in the burial requires NMR analysis. We have selected a partially solvated natively disordered fragment of Escherichia coli, thioredoxin, C37 (38-108), for full NMR spectral assignment. The secondary chemical shifts, temperature coefficients, and relaxation rates (R^sub 1^ and R^sub 2^) of this fragment indicate the presence of a flexible backbone without a stable hydrogen bond network near neutral pH. ^sup 1^H-^sup 15^N heteronuclear single quantum coherence analysis of the pH dependence of amide chemical shifts in fragment C37 within pH 2.0 and 7.0 suggests the presence of interactions between nonionizable residues and the carboxylate groups of four Asp and four Glu residues. The pH midpoints (pH^sub m^) of the amides in the ionizable residues (Asp or Glu) and, consequently, the shifts in the pH^sub m^ (ΔpH^sub m^) of these residues with respect to model tetrapeptides, are sequence-dependent; and the nonionizable residues that show pH dependence cluster around the ionizable ones. The same pH dependence has been observed in two fragments: M37 (38-73) and C73 (74-108), ruling out the participation of long-range interactions. Our studies indicate the presence of a 15-residue pH-dependent segment with the highest density of ionizable sites in the disordered ensembles of fragments C37 and M37. The observed correlations between ionizable and nonionizable residues in this segment suggest the organization of the backbone and side chains through local and medium-range interactions up to nine residues apart, in contrast to only a few interactions in fragment C73. These results agree qualitatively with the predominantly hydrophobic buried surface detected only in fragments C37 and M37 by highly sensitive differential scanning calorimetry analysis. This work offers a sensitive and rapid new tool to obtain clues about local and nonlocal interactions between ionizable and nonionizable residues in the growing family of natively disordered small proteins with full NMR assignments.
Abbreviations used: DSC, differential scanning calorimetry; ppm, parts per million; ppb, parts per billion; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser enhancement; NOESY, NOE spectroscopy; Trx, thioredoxin; C37, fragment 38-108; M37, fragment 38-73; C73, fragment 74-108.
INTRODUCTION
During the last decade a growing number of proteins variously labeled “natively unfolded” (1), “intrinsically disordered” (2,3), and “intrinsically unstructured” (4) have been found to play a key role in various biological functions that require folding/binding (5-7). For the sake of concreteness we will refer to them as “natively disordered proteins”. These proteins are easily recognized by a strong minimum around 200 nm in the far-ultraviolet circular dichroism spectrum and poorly dispersed amide proton resonances in a 1D-NMR spectrum. An intriguing question is whether these proteins have incompletely solvated regions which are essential for the folding/binding process and, most importantly, which residues are responsible for these regions. Although the presence of buried, predominantly hydrophobic regions can be easily ascertained by high-sensitivity DSC analysis, as has been done for natively disordered protein fragments (8,9), this technique lacks atomic detail, which requires the resolution provided by multidimensional NMR analysis (10-15). The latter readily provides information about the protein backbone (secondary chemical shifts; temperature coefficients; protection of amide protons to solvent exchange: NOE distance constraints; ^sup 15^N relaxation rates of the backbone; ^sup 1^H-^sup 15^N heteronuclear, etc.). However, some “natively disordered proteins” do not exhibit rigid segments in the sequence and/or regions of secondary structure (1,4), but they may still have clusters of side chains mediated by local and nonlocal interactions whose detection requires NOESY experiments. Although these experimental approaches are standard for well folded proteins, in the case of denatured or natively disordered proteins, where an ensemble of conformations is likely to be present, the analysis requires caution (12-14,16) and more laborious approaches that combine NMR with other techniques, such as mutagenesis (17), paramagnetic spin labels (18), and selective labels (19). An excellent example is furnished by the recent NOESY experiments on the selectively labeled N-terminal SH3 domain of Drosophila (drkNSH3) (19), in which folded and unfolded states coexist in slow exchange under native conditions (20-22). These experiments demonstrate the presence of at least two conformers in the natively unfolded state of drkNSH3: a compact species with native-like residual secondary structure, and a less compact one with a nonnative hydrophobic cluster mediated by long-range interactions. A methodology based on simple NMR experiments that provide clues about local and nonlocal interactions in natively disordered proteins will be a useful tool for experimentalists.
Numerous studies on folded proteins that involve NMR titration, mutagenesis, and computational analysis (23,24) have provided evidence for local and nonlocal interactions between ionizable and nonionizable residues. The pKas of carboxylate side chains in folded proteins may be shifted upfield or downfield due to the presence of hydrophobic (25,26) or polar residues in the surroundings (27-29). The pH midpoints (pH^sub m^) of the backbone amides in folded proteins reflect the electrostatic environment produced by nearby carboxylates of Asp and Glu residues (27,28,30-33). In contrast, much less is known about the pH dependence of backbone amide chemical shifts and the pKa of ionizable side chains in natively disordered polypeptides (34-41) and denatured proteins (31,42). Studies on model host-guest tetrapeptides indicate that the pHm of the amides from Asp and Glu residues are very close to the pKa of their carboxylates (32,43). Studies on natively disordered peptides and natively unfolded proteins, on the other hand, show that the pKas of the carboxylate side chains are shifted relative to the model tetrapeptides in some cases (35-37), but they are very close to them in others, even in the case of sequential carboxylate side chains (39,40). Although more studies are needed to understand the pH dependence in natively disordered polypeptides, so far these results lead us to believe that the pH dependence of backbone amide chemical shifts and carboxylate side chains may be excellent probes to identify intramolecular interactions in these polypeptides.
During the last few years, we have been studying a family of natively disordered fragments of Escherichia coli Trx as a model for biologically active natively disordered proteins (8,9,44-49). These studies have produced interesting correlations between DSC (8) and NMR experiments (48). For example, the rather rigid hydrophobic helical region of fragment N73 (1-73) correlates with the estimate of its predominantly hydrophobic buried surface. Recently, high-sensitivity DSC studies on a family of natively disordered fragments of E. coli Trx again have provided evidence for the presence of incompletely solvated regions in some fragments (9), leading us to carry out structural and dynamic NMR experiments on a fragment with a significant hydrophobic buried surface. Here we report using the pH dependence of backbone amide chemical shifts as a probe to obtain clues about local and nonlocal interactions between ionizable and nonionizable residues in the natively disordered fragment C37 and two overlapping fragments.
METHODOLOGY
Standard structural and dynamic NMR parameters of fragments
Toxic industrial chemicals as weapons: worldwide terror attacks such as the recent bomb attacks in Madrid and London have led to an increased need to be prepared for terrorist chemical threats
Toxic Industrial Chemicals (TICS) can be Chemical Warfare Agents (CWA)
Industry manufactures hundreds of TICs. Chlorine gas is found in large quantities at many water treatment facilities yet has also been used as a CWA. Phosgene gas was used extensively as a CWA in World War I, however, it continues to be used in many chemical processes. CWAs like Sarin can be simply described as powerful insecticides and similar compounds are used in the agricultural industry (like Parathion). The line between TIC and CWA often is defined only by how the chemical is utilised.
Terrorist Do Not Have to Follow the Military’s Example
The assumption often is that a chemical attack will take place in the form of military-grade chemical agent like Sarin, Soman, etc. However, it has been recognised that there are many more chemicals available to terrorists than CWAs. CWAs are tightly controlled and difficult to synthesise. Yet many toxic chemicals are present in industry and terrorists have much better access to these chemicals than to CWAs. There is no terrorist ‘rulebook’ that limits their objective.
CWA Detectors May Not ‘See’ TICs
TICs such as ammonia and chlorine are found in large quantities in virtually every community. Highly toxic chemicals like pesticides and chemical catalysts like toluene diisocyanate, are only slightly less common. Broadband sensors are not limited in their detection capabilities to a few selected CWAs, in fact, they respond to over 250 potential TICs. Surface Acoustical Wave (SAW) and Ion Mobility Spectroscopy (IMS) chemical agent detectors have algorithms with which to identify and measure specific CWAs. If a terrorist were to choose a TIC that falls outside of these algorithms, these meters are not useful The Tokyo Sarin attack was just 37 per cent Sarin, the balance being a relatively common industrial chemical, acetonitrile.
Broadband Sensor Sensivity to Chemical Agents
In February of 1999 the US Army Soldier and Biological Chemical Command (SBCCOM) released a study of Broadband sensor sensitivity to Chemical Agents entitled Testing of Commercially Available Detectors Against Chemical Warfare Agents: Summary Report. This testing showed that the Broadband sensor from RAE Systems had excellent sensitivity to HD (Mustard), GA (Tobun) and GB (Sarin) Chemical Agents.
Where Do Broadband Sensors Fit Into the Preparedness Programs?
Broadband sensors provide fast, low-level at-the-scene detection. Optional wireless integrated monitors give real time data from several monitors to the central command centre so the extent of the plume can be defined. More sophisticated monitoring equipment ie INS, GC/MS is then typically deployed for speciation purposes.
RAE System Hazardous Environment sensors
RAE Systems have been world leaders in sensing technology for over ten years.We have 18 issued and pending patents for our technology. Radiation sensors (Gamma and Neutron) have recently been introduced. Visit us at Stand DC6.
Disaster City Exhibitors
Health Effects in Army Gulf War Veterans Possibly Exposed to Chemical Munitions Destruction at Khamisiyah, Iraq: Part I. Morbidity Associated with Potential Exposure
In March 1991, U.S. troops detonated the Khamisiyah, Iraq, ammunition depot, possibly releasing two chemical warfare agents, sarin and cyclosarin. The long-term health effects associated with possible exposure to these chemical warfare agents are unknown. This study was undertaken to investigate whether possible exposure was associated with morbidity among Army Gulf War veterans using morbidity data for 5,555 Army veterans who were deployed to the Gulf region. Responses to 86 self-assessed health measures, as reported in the 1995 Department of Veterans Affairs National Health Survey of Gulf War Era Veterans, were evaluated. We found little association between potential exposure and health, after adjustment for demographic variables, and conclude that potential exposure to sarin or cyclosarin at Khamisiyah does not seem to have adversely affected self-perceived health status, as evidenced by a wide range of health measures.
Introduction
Immediately after the Gulf War, demolition was carried out in March 1991 at the Khamisiyah ammunition depot in southeastern Iraq. Troops who were possibly exposed to chemical warfare agents were identified subsequently by environmental and climatological modeling, in conjunction with unit location data for the days of the demolition.’ In this article, we compare the morbidity outcomes in the group of Army veterans possibly exposed to low levels of chemical warfare agents with those of a similar group of unexposed Army personnel. The morbidity data were collected as part of the Department of Veterans Affairs (VA) National Health Survey of Gulf War Era Veterans (NHS).2 Two other articles examine deaths associated with possible exposure3 and morbidity associated with notification of possible exposure.4
On March 4 and 10, 1991, combat engineer and explosive ordnance disposal units of the U.S. Army XVIII Corps (Airborne) destroyed two large caches of 122-mm rockets, one in a bunker and the other in a nearby pit, at the Khamisiyah ammunition supply point, -350 km southeast of Baghdad, Iraq. In October 1991, March 1992, May 1992, and May 1998, representatives from the United Nations Special Commission inspected Khamisiyah and detected the existence of sarin and cyclosarin in both intact and damaged rockets in the bunker and pit.1
Approximately contemporaneously, concerns increased about postwar morbidity among Gulf War veterans.5″12 On June 21, 1996, the Department of Defense (DoD) released a statement confirming that U.S. soldiers had destroyed ammunition bunkers at Khamisiyah, Iraq, and that one of these bunkers contained chemical warfare agents.13 Following this, the DoD made efforts to determine who was possibly exposed to chemical agents (see below) and also made efforts to notify veterans of possible exposure; the effects of these notification letters are the subject of another article.4
Toxicology of Nerve Agents Sarin and Cyclosarin
Sarin, an organophosphorus ester, is a highly toxic nerve agent. Exposure to acutely toxic concentrations can produce excessive bronchial, salivary, ocular, and intestinal secretions, as well as sweating, miosis, bronchospasm, bradycardia, muscle fasciculations, paralysis, convulsions, and death.14 Minimal effects observed at low concentrations include miosis, chest tightness, rhinorrhea, and dyspnea.14 There is limited evidence associating sarin exposure at a level sufficient to produce acute cholinergic signs with subsequent long-term health effects, such as fatigue, headache, blurred vision, post-traumatic stress disorder (PTSD), and abnormal test results of unknown clinical significance.15 At doses too low to produce acute cholinergic effects, there is insufficient evidence to determine whether there is an association with subsequent long-term health effects, in
part because of a lack of well-controlled studies.15 Cyclosarin is similar in composition to sarin, although less volatile. Its mechanism of action is similar to that of sarin, although less is known about its toxicity.15 A recent study of self-reported, long-term (25-45 years), health effects among 1,339 veterans experimentally exposed to anticholinesterase agents (including 287 exposed to sarin) included neurological and psychological outcomes such as peripheral nerve disease, vestibular dysfunction, sleep disorders, anxiety, and depression. There were only two statistically significant differences, i.e., subjects exposed to anticholinesterase agents had fewer attention problems than subjects in one control group and greater sleep disturbance problems than subjects in another control group. In contrast, self-reported exposure to hazardous chemicals outside the experimental testing program was significantly associated with all primary study outcomes.16
Methods
Study Population
The cohort for this study was selected in collaboration with the Office of the Special Assistant for Gulf War Illnesses, the Deployment Environmental Surveillance Program of the U.S. Army Center for Health Promotion and Preventive Medicine, and the VA Environmental Epidemiology Service. Eligibility for entry into the cohort was based on the veteran having served in the Gulf theater of operations. Individuals identified as having been within and outside the modeled potential hazard area were eligible for inclusion. The cohort was further defined by having participated in Phase I of the VA NHS, conducted in 1995-1997.2
The NHS was designed as a retrospective cohort study in which health factors of a population-based sample of 15,000 troops deployed into the Gulf area were compared with those of 15,000 troops serving in the military during the period of the Gulf War but not in the Gulf area. Phase I of the survey was performed in 1995-1996, before troop notification of possible chemical agent exposure at Khamisiyah, Iraq, by the DoD. A total of 11,441 military personnel, who represented four branches of service deployed to the Gulf region during the 1990-1991 Gulf War, responded to either Phase I, the postal printed questionnaire survey in 1995-1996, or Phase II, the telephone interview survey in 1996-1997. The subset of veterans who participated in the postal questionnaire survey and who served in the Army numbered 5,555.
Determining Possible Exposure
The risk factors associated with the demolition in the Khamisiyah pit in March 1991 are possible exposures to chemical warfare agents, including sarin and cyclosarin.1 For completeness, we also examined the data using exposure defined with the “50-km model,” an early exposure model that declared Gulf War veterans who were within a circle with a radius of 50 km, centered at Khamisiyah, Iraq, to have been possibly exposed.1 The history of DoD’s exposure determination efforts is given below in brief.
Determining the possible risk of chemical agent exposure to U.S. troops in the vicinity of Khamisiyah began as a joint effort by the Central Intelligence Agency and the DoD in late 1996. It quickly became apparent that the pit demolition posed a number of challenges requiring expertise in modeling the physical characteristics of open-air demolition, as well as environmental and meteorological conditions at the site. The DoD-Central Intelligence Agency team used interviews with troops who had been at the site and test demolitions and other experiments at the Dugway Proving Grounds and Edgewood Laboratories to reduce uncertainties associated with the physical and environmental conditions at the site. Because of relatively scarce meteorological data for Iraq, the team used state-of-the-art mesoscale meteorological models to simulate prevailing weather conditions over the region. Dispersion models were then used to predict the transport and spread of chemical warfare agents, based on these simulated meteorological conditions. To account for uncertainty, a conservative assumption was made to define the potential hazard area as the union of the hazard areas given by each of the various combinations of meteorological and dispersion models. The result was the generation of a potential hazard area that varied in size and shape from March 10 to March 13, 1991. From this, the team was able to determine which units of troops were presumed to have been within the potential hazard area over the course of the 4-day period. The result of this effort is known as the “1997 hazard area” (see Ref. 1 for additional details).