Rapid Headspace Oxygen Analysis

xidative degradation of drug substances in pharma-ceutical formulations is well documented (1). Althoughexact mechanistic details about what promotes reac-tions between drug substances (RH in the schematic

below) and molecular oxygen in pharmaceutical formulationsis not understood fully, such reactions are generally thought tobe in the category of auto-oxidation processes as described bythe following schematic (2):

Molecular oxygen is involved in the propagation step of this re-action, and it is an integral part of the catalytic cycle that gen-erates the oxidative degradation of drug substances in pharma-ceutical formulations.

Oxidative degradation of the active drug substance in a for-mulation decreases drug potency and reduces product shelflife (2). The rate of oxidative degradate formation is propor-tional to the structural susceptibility of the specific drug sub-stance to auto-oxidize and to storage (reaction) conditionssuch as temperature, humidity, oxygen concentration, and time.Other deleterious effects of oxidative processes include prod-uct discoloration, changes in dissolution rate and profile, pre-cipitation, and the generation of foul odors and flavors (1).Most important, oxidative degradation products generated inthe final pharmaceutical product during storage may also haveadverse pharmacological properties, including those associ-ated with toxicity or negative side effects.

Drug oxidation can also pose significant challenges duringmanufacturing processes, particularly for easily oxidized drugsubstances. The effects are especially pronounced in formula-tions that require manufacturing unit operations to be per-formed in solutions or suspensions. In such cases, extra caremay be required to minimize or eliminate exposure to oxygen.Inert-gas purging to lower solution oxygen levels as well as inertblanketing of process vessels have long been used in parenteralmanufacturing processes. Besides purging at key manufactur-ing steps — for example, during drug compounding or whileholding a drug in solution before lyophilization — packagingwith an inert blanket over the final product also protects paren-teral formulations that are susceptible to oxidation. Removing

Initiation

Propagation

Termination

RH R �

R � � O2 ROO �

ROO � � RH ROOH � R �

2ROO � molecular products

44 Pharmaceutical Technology JULY 2002 www.pharmtech.com

Rapid Headspace Oxygen Analysisfor Pharmaceutical Packaging ApplicationsAllen C. Templeton,* Yieng-Hau R. Han, Rajiv Mahajan, Rey T. Chern, and Robert A. Reed

Allen C. Templeton, PhD, is a seniorresearch chemist in the pharmaceuticalanalysis and control department at MerckResearch Laboratories, WP78-210,Sumneytown Pike, West Point, PA 19486, tel. 215.652.9204, fax 215.652.2835,[email protected]. Yieng-HauR. Han is a research associate in thepharmaceutical analysis and controldepartment; Rajiv Mahajan, PhD, is asenior research chemical engineer and ReyT. Chern, PhD, is a senior research fellowin the packaging systems developmentdepartment; and Robert A. Reed, PhD,is a director in the pharmaceutical analysisand control department, all at MerckResearch Laboratories.

*To whom all correspondence should be addressed.

OThe accuratedetermination of headspaceoxygen levels in commonpharmaceuticalpackagingconfigurations

presents several analytical challenges interms of both sampling and measurement.This article reviews headspace gaschromatography, electrochemical analysis,frequency-modulation spectroscopy,fluorescence quenching, and quadrupolemass spectrometry as methods available topackaging R&D technologists andlaboratory analysts for rapid headspaceoxygen analysis. The article describes thestrengths and limitations of each approach.

ST

EV

E S

MIT

H

Pharmaceutical Technology JULY 2002 45

one of the two key reactants in the auto-oxidation cycle fromthe package may lengthen product shelf life and ensure prod-uct quality when it reaches the consumer. Modified atmospherepackaging (MAP), a process used to package oxygen-sensitivecommercial products such as foodstuffs and medical devices inan inert atmosphere, can be used to increase shelf life. AlthoughMAP has taken hold in the parenteral pharmaceuticals indus-try, relatively few solid dosage forms are packaged under re-duced oxygen levels.

Smart packaging methods such as MAP may advance the com-mercial development of oral formulations with drug compoundsthat are particularly prone to oxidation and thus make availablecritical therapeutic agents in a high-quality, stable, and conve-nient dosage form. Integrating formulation and packaging im-provements could stabilize these compounds and shorten theirdevelopment cycle. The implementation of MAP for solid dosageforms is clearly on the horizon.

The key components to successfully using MAP for an oxygen-sensitive product are � packaging that furnishes an effective barrier to oxygen per-

meation� incorporation of an inert gas–flushing step that is compa-

tible with the speed of current product packaging lines� selection of an appropriate inert gas (e.g., argon, nitrogen, or

carbon dioxide)� rapid means to measure residual headspace oxygen content

in various pharmaceutical packaging configurations that rangefrom very low volume (tens to hundreds of microliters) torelatively high volume (milliliters to liters).Fast, sensitive methods for monitoring headspace oxygen

levels would greatly benefit the development and use of MAPand facilitate its implementation in packaging R&D, manu-facturing process development, and final product quality assur-ance. For packaging R&D, robust oxygen measurement meth-ods would enable real-time screening of the suitability ofpackaging systems (all components of a container and closure)for MAP packaging applications. The most obvious exampleis the determination of oxygen ingress kinetics for a packag-ing system under both realistic and accelerated storage condi-tions. Experimental data could be used to develop predictivemodels for each critical packaging system component to sup-port future MAP development programs.

For manufacturing process development, rapid and accurateoxygen determination in the development feedback loop wouldbenefit the scale-up and validation of a manufacturing processthat implements maximum package throughput per unit timewhile achieving and maintaining critical threshold levels foroxygen. Tools that provide quality assurance and quality con-trol information at-, on-, or off-line would ensure that the oxy-gen content of the inert atmosphere in the packaging containerheadspace is below the predetermined specification for the prod-uct. Such testing also could assess the container-closure integrityand could augment or replace current microbial ingress mea-surement methods that are based on sterility assessment.

The rapid and accurate determination of headspace oxygenlevels in the various packaging forms used in the pharmaceu-tical industry poses several unique analytical challenges that

are beyond the capabilities of a single measurement technique.This article describes various measurement options that areavailable to packaging R&D technologists and laboratory an-alysts in the context of pharmaceutical packaging applications.The article is organized by technique, with a brief introduc-tion to measurement principles followed by a discussion ofpharmaceutical packaging applications.

ExperimentalSample preparation. Standards that contained various concen-trations of oxygen in nitrogen were prepared from double gravi-metrically certified gas cylinders (Scott Specialty Gases, Plum-steadville, PA). The concentrations were 0.00, 5.0, 10.0, 15.0,and 20.0% oxygen in nitrogen. Vials filled with air (20.9% oxy-gen) were substituted in some experiments for a 20.0% stan-dard. In brief, gas from each respective cylinder was fed slowlyinto a portable glove bag (Spilfyter, Thomas Scientific, Swedes-boro, NJ) by means of a short length of Tygon tubing. Afterpurging for �30 min to remove residual atmosphere (longerpurge times provided no benefit), 10-mL clear glass vials (25� 54 mm Type I untreated, Kimble/Kontes, Vineland, NJ) eachwere filled under a given atmosphere, and 20-mm gray rubberstoppers (S-87J, 4405/50, West Pharmaceutical Services, Li-onville, PA) immediately were positioned into the vials. Afternearly 50 vials were filled, they were removed from the bag,and an aluminum flip-top cap was crimped into place on eachvial. Using an oxygen–carbon dioxide analyzer (CheckMate9900, PBI Dansenor A/S, Ringstëd, Denmark), vials from eachprepared lot were analyzed immediately and after three months’storage in ambient laboratory conditions (19–22 �C, 45–55%RH) for changes in relative oxygen levels. Comparisons of thetwo timepoints confirmed that no oxygen had leaked into thevials. In fact, this vial–closure combination, which is used forcommercial parenteral pharmaceutical products, has beenshown to maintain headspace integrity for at least two years.

Although the previously described sample preparationmethod undoubtedly fails to produce the exact concentrationsof gases in the prepared vials as those provided in the corre-sponding certified cylinders, the use of these vials for relativecomparisons of the capabilities of each technique should bevalid. For the purposes of the experiments, the vial contents(vial-to-vial variability in each lot was found to be very low)are understood to be the same as those of the respective filltanks. Of course, any attempt at an absolute measurement ofvial oxygen contents with any given analytical method will besubject to the same technique biases that this report attemptsto address. To facilitate comparison, for a given concentration,vials from the same lot of prepared samples were used for com-parative purposes for all five measurement techniques. For ex-ample, of the nearly 50 vials prepared at 5.0% oxygen levels,five were reserved for measurement for all five techniques ex-amined in the study. Measurement of relative vial-to-vial con-tent variability using the CheckMate 9900 oxygen–carbon diox-ide analyzer indicated excellent content uniformity (nodifferences) among five randomly pulled vials.

Instrumentation. Headspace gas chromatography (GC).Headspace GCwas implemented using a GC system (HP 6890, Agilent Tech-


nologies, Wilmington, DE) equipped with a thermal conduc-tivity detector. Separations were performed using an HP porouslayer open tubular (PLOT) Molesieve 5-Å column (30 m long� 530 �m i.d.) with 50-�m film thickness (Agilent). The thin-film HP PLOT Molesieve 5-Å capillary column is a fused-silica column coated with a homogeneous layer of 50-�m-thick5-Å molecular-sieve zeolite stationary phase. Because of thecolumn’s sensitivity to moisture, a moisture trap was installedin-line with the carrier-gas feed. Injections were performedwith a 25-�L gas-tight locking syringe (Hamilton, Reno, NV)fitted with a 22S-gauge needle with a #2 point style. Chroma-tographic conditions were as follows:� carrier gas: helium (2 mL/min)� oven temperature: 26 �C � inlet: 160 �C, split mode, 10:1 split ratio, 20 mL/min split flow� injector: 160 �C� run time: 10 min� TCD detector: 160 �C.A series of standards and system-suitability samples also wasanalyzed.

Electrochemical methods. The authors used the CheckMate 9900oxygen–carbon dioxide analyzer equipped with a solid-statezirconia ion-selective electrode for oxygen determination anda nondispersive infrared spectrometer for carbon dioxide mea-surements. The instrument was warmed up for 10 min beforemeasurements were taken and was calibrated according to thevendor’s specification. The instrument was set to withdraw 2 mL of headspace sample using a small internal-diaphragmpump that fed the sample into a small cell that contained themeasurement electrode. The carbon dioxide measurement mod-ule was turned off during the experiments.

Frequency modulation spectroscopy. A headspace oxygen analyzer(FMS-760, Lighthouse Instruments, Charlottesville,VA) equippedwith a tunable diode laser source (762 nm) and photodiode de-tector was used for frequency modulation spectroscopic mea-

surements. The instrument was allowed to equi-librate for 30 min before taking measurements.The sample holder was purged constantly withdry nitrogen set at a flow rate of 3 standardL/min, and measurements were acquired at asampling rate of 100 Hz.

Fluorescence quenching. A miniature spectrom-eter (SF2000, Ocean Optics, Inc., Dunedin, FL)equipped with a 300-�m bifurcated opticalfiber and a 21-gauge, custom-designed needleprobe was used for oxygen analysis. The cus-tom needle probe consisted of a 2-mm shaftopening offset by �2 mm from the pointed tipof the needle. The blistal end of the aluminum-clad bifurcated optical fiber was fed into theneedle and exposed at the shaft opening. Theexposed optical fiber tip was coated with a fluo-rophore sol-gel film. Fluorescence intensity asa function of oxygen concentration was moni-tored by means of the same fiber at a charge-coupled device 2048-element silicon detectorarray.

Quadrupole mass spectrometry. A quadrupole mass spectrometerequipped with a miniature array of nine quadrupole mass ana-lyzers (Micropole, Ferran Scientific, San Diego, CA) was used foroxygen determination. The system’s roughing and turbomole-cular pumps achieved low base vacuum pressures (<10�7 Torr).A thoriated iridium electron source ionized the sample beforequadrupole mass filtering. The ions formed at the source wereextracted and focused into the entrance apertures of the quadru-pole array by a series of lenses. Detection was achieved at a Fara-day cup positioned at the opposite end of the quadrupole array.The instrument was operated in a trend gas–only mode whereinthe analysis residence time per each gas monitored was set to 4.5 s, and the partial pressures of each monitored gas wererecorded during the course of the measurement. An analog massspectra output that allows for species identification throughoutthe mass range (2–300 amu) of the detector was used.

Headspace GCPrinciples of operation. When a problem arises in a pharmaceu-tical research laboratory that requires analysis of a headspacesample, headspace GC is probably the first method that comesto mind for solving the problem. This approach is reasonablegiven that a vast body of knowledge, stretching back 40 years,describes its theory and application (3). Although headspaceGC is an analytical problem-solving tool with capabilities farbeyond the bounds of oxygen analysis, other techniques canprovide similar or better results for specific headspace analytes,often in a more timely and simplistic fashion. The followingdescription of headspace GC is intended only as a cursoryoverview. The authors refer those who are interested to the sub-stantive literature about the topic for greater detail (3).

GC is inherently a technique for the study of volatile com-pounds. Figure 1 is a schematic of the components that con-stitute a typical GC instrument that is equipped with a splitinjection port and a capillary column. For headspace sampling,

Two-stagepressureregulator

Soap-bubble meter

SyringeDetector

Recorder

Electrometeror bridge

A/D

Datasystem

Injector

Flow splitter

Column

Column oven

Rotometer

Flowcontroller

Carrier-gas supply

Figure 1: The components of a typical GC instrument.


a gaseous sample is introduced by injection into an inert mov-ing gas stream called the mobile phase or carrier gas.

The mobile phase transports the sample onto a stationaryphase where the components of the sample are separated bymeans of selective partitioning between stationary and mobilephases. The time, referred to as retention time (tR), that elapsesbetween sample introduction and the point at which peaks ap-pear at the detector is characteristic of the properties of the ana-lyte molecules. The area of the respective peaks as they appearin the resultant chromatogram is proportional to the analyteconcentration in the sample, as related by a measurable responsefactor. As applied to the analysis of oxygen in pharmaceuticalpackaging applications, a sample of headspace (tens of micro-liters) is removed from a package by withdrawing a portion ofsample with either a gas-tight syringe or a GC autosampler topressurize the container with mobile phase to displace an aliquotof sample. Performing the experiment in either manner requiresoperation in what is referred to as the static mode of headspacesampling, wherein the goal is to sample the equilibrium con-tents of a package headspace at a given instance.

Figure 2, which shows an example chromatogram of lab air,illustrates the determination of headspace oxygen by means ofa megabore capillary column with thermal conductivity detec-tion. Specific experimental conditions for the analysis are de-scribed in detail in the “Experimental” section of this article.As in any type of chromatographic analysis, selection of the ap-propriate column to achieve separation of the sample gas mix-ture into its various components is pivotal.

To separate oxygen from argon and nitrogen in an air sampleduring a 10-min analysis, the authors used a commercially avail-able thin-film PLOT capillary column consisting of a fused-silica capillary tube coated with a homogeneous 50-�m-thickzeolite molecular sieve (5-Å pore size) film. The mode of sepa-ration is based on the size of the analyte molecule or atom;retention time is as follows: nitrogen > oxygen > argon. The netarea and area percentage of oxygen are determined using thefollowing equations

in which x is argon, nitrogen, or oxygen; AreaRaw,x is raw peakarea of x (mV s); AreaNet,x is net peak area of x after conversion;�He is the conductivity of helium (154.6 W/m K); and �x is theconductivity of argon (17.8 W/m K), nitrogen (25.9 W/m K),or oxygen (26.2 W/m K) (4).

The authors evaluated the linearity of headspace GC for thedetermination of oxygen in the range of 0.00–20.9%. A gas-tight locking syringe was used to extract and manually inject25 �L from the contents of vials that contained 0.00, 5.0, 10.0,or 15.0% oxygen, and lab air (see the “Experimental” sectionfor details about sample preparation). As previously mentioned,

AreaNet, x % AreaNet, x

AreaNet, Ar � AreaNet, N � AreaNet, O

� 100%

AreaNet, x �He � �x

�He

� AreaRaw, x

for a given concentration, vials from the same lot of preparedsamples were used for comparative purposes for all five tech-niques discussed in this report. The technique displayed goodlinearity throughout the concentration range explored (r2 0.9903; data not shown), but a substantial y offset was observedthat was studied in subsequent experiments. A series of experi-ments was performed to examine � amount of leakage of the package during sample removal � syringe leakage during sample transport from package to in-

strument� septum port leakage during sample introduction.For the type of sample studied, all three sources showed neg-ligible amounts of sample contamination.

Another source of bias introduction that was examined wasresidual air from the syringe barrel. Three sizes of syringe needles were used, and a direct correlation between bias and syringe-barrel volume was observed. Purging the syringe-needle volume with carrier gas before sampling reduced biasfrom 4689.9 area counts by approximately tenfold, giving 0.08%for the 0.00% oxygen standard. Precision was evaluated by per-forming five replicate analyses (with a 22S-gauge needle, nopurging) of 20.9% (air, high) and 0.00% standards (low); theseexperiments yielded an average of 22.1% and 0.83% with a relative standard deviation (RSD) of 1.6% and 10%, respec-tively. The limit of quantitation (LOQ) (signal to noise [S/N]10) of oxygen for the technique was estimated at �0.25%without purge and 0.025% with purge step. However, samplingbias must be understood for any specific system before accu-rate data at low levels can be realized.

Applications. The use of headspace GC to analyze oxygen inpharmaceutical packaging configurations offers several ad-vantages such as the widespread availability of instruments anduser familiarity. For sample containers of appropriate dimen-sions, the autosampling capability of commercial headspaceGCs makes the instrument particularly appealing for analyz-ing samples en masse. GC autosampler vial dimensions typi-cally are not consistent with common pharmaceutical packagesizes, thus the use of an autosampler usually is limited to either a few samples of appropriate dimension or samples thatcan be transferred to headspace autosampling vials. The latterwould defeat the purposes of many of the investigations described in this article.

Withdrawing and injecting samples manually with a gas-tightsyringe can pose various problems. For example, when work-ing with samples that are under vacuum, pressure equalizationwith ambient air can lead to sample dilution and erroneouslylarge values for oxygen. Therefore, one must use a gas-tightlocking syringe and institute appropriate controls as describedin the previous discussion. Moreover, one must carefully re-move samples from packages to avoid introducing ambient at-mospheric leakage during the sampling process. Our experi-ence showed that applying a self-adhesive rubber septum beforepuncturing the sample usually was sufficient as a preven-tative measure when puncturing aluminum foil (e.g., foil induction–sealed high-density polyethylene [HDPE] bottles).Sealing samples with rubber stoppers (e.g., lyo vials) usuallyprovides an adequate barrier to leakage during sampling. Sam-


pling errors are common to all destructive headspace oxygenanalysis methods, and they demonstrate the difficulties of quan-titatively measuring small amounts of an oxygen analyte with-out contaminating it with air. Despite problems with sampleremoval, the low absolute sample volume requirements (tensof microliters) make static headspace GC an attractive and po-tentially useful analysis tool for pharmaceutical packaging formsof all types and sizes.

In addition to sample introduction, the major limitations ofthis technique for the analysis of oxygen are the requirement ofa relatively lengthy separation step (a few minutes at best) to beoxygen selective, the need to assess appropriate standards, andthe need to demonstrate system suitability throughout the analy-sis of a sample set. Although traditionally a GC instrument hasa large footprint that would make at-line work unfeasible, therecent commercial development of portable GC units (some-times termed micro-GCs) is beginning to make at-line sam-pling more feasible.

Electrochemical methodsPrinciples of operation. The use of electrochemical methods foroxygen analysis also has long historical precedence from botha scientific and a commercial perspective. The basis of opera-tion of most commercial electrochemical headspace-oxygenanalyzers is a solid-state zirconium oxide ion-selective elec-trode (5). With this type of sensor, oxygen ions migrate intodefect sites in the ceramic lattice structure of zirconia at ele-vated temperatures (>400 �C). As has been known since thetime of Nernst (1899), when gases of differing concentrationsreside on opposite sides of an electrode membrane, a measur-able potential (E) difference is generated. If one side of themembrane is exposed to gas of reference-oxygen concentra-tion ([O2]REF), then the potential generated in the presence ofa gas of unknown oxygen concentration ([O2]UNK) is propor-tional to the difference in oxygen concentration across themembrane, as shown by the Nernst Equation

in which E is potential (V), R is the gas constant (J/K), Tis the temperature (K), n is the number of electrons trans-ferred, and F is the Faraday constant (J/V) (5).

In practice, the measurement procedure involves punc-turing the package and removing 2–4 mL of headspace forpurging and subsequent analysis in a small cell that con-tains the electrode. The analysis is rapid, typically requir-ing �5 s. The measurement is not adversely affected by hu-midity, but the sensor can be damaged if it is inadvertentlyexposed to liquid samples. Some commercial instrumentssimultaneously measure oxygen and carbon dioxide levelsin headspace samples and are available with sampling acces-sories for a variety of package containers (e.g., aluminumcans, beer bottles). Other commercial electrochemical oxygen-analysis techniques are based on the direct reduc-tion of oxygen at a planar gold electrode (Clark techniquesor modifications thereof), but the authors found these tech-

niques to offer no advantage compared with those that are de-scribed in this article, thus they are not discussed.

In the present study, a single-point calibration of the instru-ment in laboratory air was performed (yearly calibration typi-cally is advocated), followed by immediate analysis of a set ofprepared standards (see the “Experimental” section). The lin-earity of the analytical method was evaluated for oxygen overthe range of 0.00–20.9%, and the technique was found to de-liver good linearity (r2 0.9984; data not shown). The authorsevaluated precision by performing five replicate analyses of20.9% (air, high) and 0.00% standards (low). These experimentsyielded an average of 20.6% and 0.11% with an RSD of 0.00%and 13%, respectively. The vendor-indicated LOQ for the par-ticular instrument was 0.0001% (1 ppm). As with GC, samplingerrors may contribute to the observed bias, as discussed laterin this article.

Applications. The sample-volume requirements of electro-chemical methods limit the use of this analysis approach tolarge-dimension pharmaceutical packaging types with �2-mLheadspace volume such as HDPE bottles, glass vials, and blis-ter packages (individual blisters) of sufficient volume. Therugged character of such instruments and their ease of use makethe technique particularly suited for the manufacturing setting.

Frequency-modulation spectroscopy (FMS)Principle of operation. Recently commercialized for package in-spection applications, FMS is a high-sensitivity laser absorp-tion technique for nondestructive monitoring of gas concen-trations in optically transparent containers (6). Oxygen absorbsnear-infrared (NIR) light in a band of rotational transitionscentered at 762 nm. Oxygen absorbance measurements at thiswavelength with standard spectrometers that use incoherentwhite-light sources lack the sensitivity to provide a useful mea-sure of headspace oxygen levels because of low-frequency lampintensity fluctuations. However, the measurement of S/N canbe improved vastly (100–10,000 �) with laser-light sources and

E RTnF

lnO2 REF

O2 UNK240

200

160

120

80

40

Inte

nsity

(m

V)

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

Time (min)

Oxygen

Nitrogen

Argon

Figure 2: An example chromatogram illustrating the determination ofheadspace oxygen by GC with a PLOT molecular sieve column with thermalconductivity detection.


frequency modulation detection techniques. When a tunablediode laser is modulated with a radio frequency oscillator, thedetection bandwidth can be shifted to high frequency at whichintensity fluctuations, inherent to low-frequency measurements,are minimized.

The measurement is conducted by frequency modulating adiode laser by superimposing a radio frequency oscillation, �,onto the diode-injection current. The spectral output of a frequency-modulated diode laser, shown at the top of Figure3, consists of a carrier frequency, c, and side-band frequen-

cies, c � �. When the laser is scanned throughthe wavelength of oxygen absorbance, the amountof light absorption, which is proportional to thegas concentration, is “written” into the side-bandfrequencies by recording the difference in absorp-tion between the two side bands. The differentialabsorption information is recovered by means ofphase-sensitive detection techniques. Figure 4shows the demodulated absorption line shape. Theamount of light that is absorbed is directly pro-portional to oxygen concentration. The gas den-sity, n, is related to the peak-to-peak signal am-plitude, �I, according to Beer’s law, which for aweakly absorbing molecule is shown by

in which �� is the change in intensity of light afterpassing through the container (W/cm2), � is thefull width at half maximum of the absorption sig-nal (cm�1), � is a constant, Io is the incident laserintensity (W/cm2), S is the integrated absorptioncross section (cm2 � cm�1), and x is the containerdiameter (cm). The measured density in a samplevial is referenced to a standard and displayed as aconcentration in percentage.

The authors investigated the capability of thetechnique to measure oxygen in 10-mL clear glass vials. A single-point calibration of a factory-supplied 20% oxygen stan-dard was used to calibrate the instrument. The linearity of theanalytical approach was evaluated for oxygen over the rangeof 0.00–20.0% by analysis of 0.00, 5.0, 10.0, 15.0, and 20.0%standards. Each sample was measured by placing a vial in asmall optical sample holder and collecting absorption data foreach sample for 10 s at a sampling frequency of 100 Hz. Thetechnique displayed good linearity in the range of concentra-tions examined in the study (r2 0.9948; data not shown).Precision for the technique was evaluated by performing fivereplicate analyses of 20.0% (high) and 0.00% (low) standards.These experiments yielded an average of 19.8% and 0.19% withan RSD of 0.05% and 26%, respectively. Because the instru-ment is nondestructive, the precision of replicate analyses onthe same vial also was evaluated by measuring, removing, re-inserting, and remeasuring the sample. Five repeat measure-ments on a single 20.0% and 0.00% standard indicated goodprecision for the method, with an RSD of 1% and 34%, respec-tively. The vendor-specified quantification limits for the mea-surement were estimated at 0.38% oxygen with a S/N of 2 and1.9% oxygen with a S/N of 10.

Applications. The application of this technique is limited toheadspace monitoring in packages that are optically transpar-ent (e.g., glass vials, ampuls, and bottles, including colored glass-ware such as amber) or translucent (low-density polyeythylenebut not HDPE). Other containers with various optical densi-ties and corresponding transmittance properties also can besampled but must be evaluated on a case-by-case basis. Pack-

n ��•�•�

I o•S •xcm�3

Recorder

Datasystem

Current andtemperature

controller

Scancontroller

rfoscillator

A/D converter

Computer

Amp

MixerLo RF

IF

DetectorSampleDiodelaser

(a)

(b)

c c c

c � � c � �

c � � c � �

Figure 3: a) Frequency and intensity profile of a diode laser beam, from left to right:unmodulated, modulated with no absorption, and modulated with absorption by theupper sideband. b) Schematic diagram of an instrument for frequency modulationspectroscopy. The frequency modulated diode laser output is converted to anamplitude modulation after passing through a gas sample which absorbs at aparticular wavelength. The amplitude modulation is proportional to gas concentrationand can be phase sensitively detected and related to oxygen concentration.

0.4

0.2

0.0

�0.2

�0.4

�0.6

�0.8

�1.0

�1.2

�1.4�1.6

Inst

rum

ent r

espo

nse

(V)

Wavelength (arbitrary units)

20%

13%

8%

4%

2%1%0%

Figure 4: Frequency-modulation signals from oxygen absorption. Thepeak-to-peak amplitude of each spectra is proportional to oxygenconcentration (noted to the right of each scan).


ages are inserted into a sample holder that can be customizedfor cell volumes of �1–100 mL, which typically present cellpath lengths of 1–3 cm.

The method’s principal advantage is that it can nondestruc-tively analyze oxygen contents within sealed containers withoutchanging the sample. Thus, a single package can be used to studyoxygen concentration profiles at periodic intervals and in variousstorage conditions without the need to destructively insert a probeor remove the headspace contents for analysis. The value of thisadvantage cannot be overstated in terms of absolute sample re-quirements, sample preparation time, and sample-to-sample varia-bility. From a quality assurance perspective, substantial cost sav-ings can also be realized from a nondestructive analysis approach.

Fluorescence-quenching methodsPrinciples of operation. Oxygen-sensing technologies that are basedon luminescence quenching are also a relatively new com-mercial development, and the scientific underpinnings for theiroperation are described extensively in a recent review article (7).This discussion will introduce briefly this oxygen measurementtechnology; the reader is referred to more-specialized literaturefor further detail. Figure 5 shows a commercial configuration inwhich fluorescence is used to measure the partial pressure ofoxygen by using a bifurcated optical fiber to transmit an excita-tion source from a blue–green LED (�max 470 nm) to a thin-film coating applied to the tip of the fiber. A ruthenium com-plex dispersed in the thin film is excited and produces afluorescence emission maximum at 600 nm that is collected atthe tip and carried back to a charge-coupled device detector. Theintensity of fluorescence observed is inversely proportional tothe amount of oxygen in a liquid or gaseous sample.

Luminescence-quenching sensors can be explained by thefollowing processes in which M represents the luminescingmolecule and Q is a quencher:

Photon absorption (Ia)

Luminescence (kr)

Nonradiative decay (knr)

Dynamic quenching (kq)

M � h� M*

M* M � h�

M* M � �

M* � Q M � Q*

Quenchers act by depleting the excited-state population (M*),thus lowering the intensity of luminescent emission or shorten-ing decay time. In the case of commercial oxygen sensors, M isa fluorescent ruthenium dye whose fluorescence intensity isquenched by oxygen in a manner commonly referred to as dy-namic fluorescence quenching. In terms of the process previouslydescribed, a collision of an oxygen molecule (Q) with the fluo-rophore in its excited state (M*) leads to a nonradiative energytransfer and a loss of fluorescence intensity.

The amount of fluorescence-intensity quenching can be quan-titatively related to the partial pressure of oxygen in a samplefrom the simplified Stern–Volmer equation (8)

in which I is the quenched fluorescence signal intensity, Io is theunquenched fluorescence signal intensity, K is the quenchingconstant that relates to a particular fluorophore used as M, andpO2 is the oxygen partial pressure.

An expanded version of the Stern–Volmer equation con-taining a factorial expansion of these same terms can also beused to relate Io to oxygen levels in a more exact manner. Stan-dard operation includes, at minimum, a daily calibration at sev-eral oxygen concentrations at a fixed pressure and temperatureand use of the measured calibration coefficients to determinethe concentration of oxygen in an unknown sample.

In the current study, an instrument that is based on fluorescence-quenching technology was calibrated with six oxy-gen concentrations (see “Experimental” section) before initiat-ing analysis. Although a single-point calibration is recom-mended, the authors have found that this practice is insufficientto provide reasonable accuracy over a 0.00–20.9% oxygen con-centration range. Moreover, a second-order polynomial fit ofthe data is required to generate calibration coefficients acrosswide ranges of sample concentrations (more than a few per-cent). Also, frequent recalibration is necessary because the sen-sors tend to degrade under normal operating conditions. Thischaracteristic is particularly true of new sensors, which may re-quire “burn in” for a few hours before stable operation isachieved. Analyses were accomplished by inserting a customneedle probe (see “Experimental” section) into a 10-mL sam-ple vial and waiting 1 min for equilibration of the sensor to theheadspace environment. Shorter equilibration times can be usedand are a function of the chemical composition of the protec-tive overcoat applied to the sensor tip to exclude ambient light(a PTFE coating was used in this study).

The linearity of the analytical approach was evaluated foroxygen over a 0.00–20.9% range in the present study. The fluorescence-quenching technique demonstrated a linear rela-tion between measured versus standard concentration (r2 0.9952; data not shown), but an offset in the data predicted�0.83% oxygen for the 0.00% standard, which measured at1.6%. The accuracy of fluorescense quenching is commonlyknown to be limited by resolution (random noise), deviationsfrom the Stern–Volmer relationship, and calibration error (8).Which, if any, of these factors may have promoted inaccuracyin the previously described experiments has yet to be experi-

I o

I 1 � K pO2

Thin sol-gelfilm containingRu complex

Linear CCD-arraysilicon detector

20 mA blue—green LED�max 470 nm

Bifurcated optical fiber

�ex 470

�em 600 nm

Figure 5: Schematic diagram of a headspace oxygen analyzer basedon fluorescence quenching.


mentally isolated. Measurement precision was evaluated by per-forming five replicate analyses of 20.9% (air, high) and 0.00%(low) standards; these experiments yielded an average of 20.3%and 1.6% with an RSD of 1% and 17%, respectively.

Applications. Fluorescence-quenching sensors do not actuallyconsume headspace oxygen and thus can be miniaturized foruse on very small volume samples. Still, the technique is de-structive because the tip of a 300-�m probe must be used topuncture the package and come in contact with the relevantheadspace, allowing an effective smallest volume of �100 �Lto be probed.

Some attributes of the technique include � rapid response times (from 5 s to 2 min) that are limited to

the time required for equilibrium to be reached between oxy-gen in the sample headspace and the sensing film by way ofdiffusion through the protective overcoat

� very low sample volume requirements (�100 �L) that arelimited only by the contact dimensions of the probe

� low cost and small instrument footprint.The low sample volume requirement makes the fluorescence-

quenching sensor the only instrument besides headspace GCthat is capable of rapidly measuring oxygen levels in individualblister cavities of typical dimensions used in pharmaceuticalpackaging. In addition, the sensor’s small size and easy setupmake it very attractive from a portability perspective for achiev-ing at-line packaging measurements. Fluorescence-quenchinginstruments are also available in multichannel configurationsthat allow the simultaneous measurement of several samples,and the data-logging component of the software executes pro-grammed measurements at fixed time intervals.

The fluorescence-quenching method does have some limita-tions, however. Care must be taken to calibrate throughout theintended measurement temperature range of samples becausefluorescence-quenching levels change as a function of tempera-ture. Temperature affects the measurement by modifying fluo-rescence decay time and the collisional frequency of oxygen withthe fluorophore. Requirements to maintain the sample at �1 �Cor to concurrently measure temperature while conducting themeasurement curtail the practical utility of the method. Expe-

rience in our lab also has indicated a need to consistently uselonger signal-integration times during the life of any given probeto achieve sufficient signal intensities (1800 counts over the mea-surement time at zero oxygen) over background noise. The de-creased response factor that was observed could be the result ofchanges in the ruthenium dye (e.g., degradation) over time.Higher signal intensities or lower noise levels are desirable be-cause S/N is proportional to the square root of integration time.Moreover, as can be seen from the Stern–Volmer equation, therate of change of signal intensity with quenching is highest atlower oxygen levels. Further development of the instrumenta-tion for oxygen-specific measurements would benefit greatly theoverall utility of the technique.

Quadrupole mass spectrometric analyzersPrinciples of operation. Quadrupole mass analyzers are by far themost common type of mass spectrometer in use today, and theliterature about this type of analyzer is extensive (8). Quadru-pole mass analyzers often are thought of as mass filters becausethey can be tuned to transmit ions of a narrow range of mass-to-charge (m/z) ratios. Figure 6 shows a generalized blockschematic of a quadrupole mass spectrometer. A typical quadru-pole instrument separates ions with differing masses by apply-ing a combination of static- and radio-frequency electric fieldsto four cylindrical rods. A headspace gas sample is introducedat an inlet and fed into an ion source where electrons are emit-ted from a filament and ionize the sample gas. The sample ionsthen are accelerated in an electrical field and are injected intothe opening at the center of the rods. In the most simple sys-tems, one pair of rods is connected and attached to the positiveterminal of a dc power source, and the other connected pair isattached to the negative end of the same source. A variable rffield with phase differences of 180� also is applied to each con-nected pair of rods with an ac power supply. For an ion to travelthrough the quadrupole to the detector, the ion must have a stable trajectory in the presence of these applied voltages andelectric field, as described by the Mathieu equations of motionfor ions. The application of a particular set of voltages to therods allows mass filtering and the transmission of a band of ionswith a limited range of m/z values. This process usually is accom-plished by scanning the rf voltage and dc voltages from zero toa predetermined maximum value while maintaining a fixed ratio.In this manner, a mass spectrum of analyte ions reaching thedetector can be recorded at a Faraday-cup detector.

The quadrupole mass analyzer used in this study compriseda set of 16 identical cylindrical rods arranged to form ninequadrupoles (8). The mass resolution of the quadrupole was�1 amu, and the mass range of the analyzer was 2–300 amu.The sample was introduced to the instrument by means of acustom-made analysis port with a toggle switch to introducesample from a needle used to puncture a sample package. How-ever, other package-puncturing sampling techniques could alsobe used (e.g., syringe and injection port). The linearity of theanalytical approach was demonstrated for oxygen over the rangeof 0.00–20.9% (r2 0.9965; data not shown). Precision wasevaluated by performing five replicate analyses of 20.9% (air,high) and 0.00% (low) standards; these experiments yielded an

Ion with unstable trajectory

Ion withstable trajectory

dc and rf voltagesIonizing

electron beam

Ioncollector

Figure 6: Block schematic of a quadrupole mass spectrometer.


average of 21.9% and 0.00% with an RSD of 0.5% and 2.6%,respectively. The very small amount of offset observed for thisinstrument was the result of the extremely low LOQ (estimatedat sub-ppm levels) of the measurement technique, limited onlyby the lower limit of vacuum achieved in the analyzer.

Applications. The application of quadrupole mass analyzersin a residual gas–analysis mode has long precedence in thesemiconductor industry in which residual contaminant gaslevels in various ultrahigh vacuum chambers must be moni-tored and controlled. The principal advantage of using quadru-pole mass spectrometry for pharmaceutical packaging appli-cations is that many analytes can be monitored simultaneouslyand quantitated (2–300 m/z) with one injection of headspacesample. As is the case for other destructive techniques, thelargest drawback for this instrumentation is sample introduc-tion. Several sample-introduction strategies are being exploredto improve the utility of the approach. The technique likelywould be useful in process-monitoring applications in whichthe detailed contents of an inert gas blanket beyond that ofsimple oxygen levels is required.

Which technique should I use?The answer to this important question depends on the type ofinformation desired, instrumentation availability, and the typeof packaging being considered. In MAP development, the stud-ies may comprise research into the oxygen permeability prop-erties of various packaging systems, packaging-system selec-tion, process development, process implementation, and finally,quality assurance. For the evaluation of various packaging sys-tems, the most common questions may be about understand-ing the rates of oxygen ingress (or consumption) ranging fromvery low levels (�1% oxygen) to ambient oxygen concentra-tions. The types of tools used in a research laboratory shouldbe reevaluated when considering a manufacturing setting forprocess development and implementation. In addition, in the

QA/QC setting other con-siderations such as long-term method robustness,stability, ease of calibrationand use, oxygen levels to beroutinely analyzed, andother practical considera-tions will help guide tool se-lection. The forthcomingdiscussion will examine toolselection criteria in terms ofperformance considerations,other relevant technique at-tributes, and type of pack-age being considered.

First, what can we learnabout how the five tech-niques presented in this ar-ticle performed in terms ofmeasuring oxygen concen-trations in the range of zeroto roughly ambient oxygen

levels? The data shown in Table I are clarified in the followingdiscussion of the performance parameters that were studied.

Key points. Linearity. All five techniques that were investigateddisplayed good linearity over the measurement range0.00–20.0% or 0.00–20.9%, all displaying r2 �0.9903.

Measurement precision. The standard deviation and RSD at bothhigh (20.0%) and low (0.00%) ends were examined for eachtechnique. As the data show, the precision for the techniques atlow concentrations ranged from 2.6% to 26%. Note that theRSD of five replicate measurements at ambient oxygen levelswere �2% for all five of the techniques.

Accuracy. Generally speaking, for most MAP programs the aimis to reduce oxygen values from ambient to a predeterminedspecified level. Often, the measurement technique may not berequired to give the absolute oxygen level in the container butonly to be sufficiently accurate to indicate that the oxygen con-tent of the package is below the target threshold, thus servingas a limit test. Thus, the importance of the measurement tech-nique to measure at low levels (e.g., �5%) is more importantthan capabilities at high levels. One way to interpret the per-formance data in Table I in this context is to examine the ab-solute differences observed between theory and experiment forthe low-level oxygen standard. As Table I shows, the degree ofdeviation (from least to greatest) that was observed betweenpredicted and measured low oxygen level standards followedthis trend: quadrupole MS < electrochemical < FMS < GC <fluorescence quenching.

LOQ. The lower LOQs shown in Table I were not measured di-rectly as part of the study. In instances in which they are pro-vided, the values are either estimated or obtained from the ven-dor. For critical absolute oxygen concentration measurementsat very low levels, the lower LOQs should be established for thespecific system being studied. Clearly, the sampling methodplays a critical role in the quality of the data for oxygen levels�2% and limits the accuracy of the data.

Table I: Performance parameters for the oxygen-analysis methods evaluated.Standard Measured Precision Accuracy Linearity LOQ

Method (%)* (%)** Std. Dev.† RSD (%) Bias‡ r 2 (%)§

Headspace GC 20.9 22.1 0.30 1.6 1.20 0.9903 0.250.00 0.83 0.08 10.0 0.83

Electrochemical 20.9 20.6 0.00 0.0 �0.30 0.9984 0.0001**0.00 0.11 0.01 13.0 0.11

FMS 20.0 19.8 0.01 0.05 �0.20 0.9948 1.90.00 0.19 0.05 26.0 0.19

Fluorescence 20.9 20.3 0.20 1.0 �0.6 0.9952 unknownquenching 0.00 1.6 0.27 17.0 1.6

Quadrupole MS 20.9 21.9 0.10 0.50 1.0 0.9965 sub-ppm0.00 0.00 7.54 � 10�5 2.60 2.9 � 10�3

* Prepared as described in the “Experimental” section of this article.** Average of five different samples.

† Standard deviation.‡ Bias calculated as measured—theoretical.§ Limit of quantitation, S/N 10, all specified by instrument supplier with the exception of headspace

GC, which was measured.


Knowing the performance limitations of a particular in-strument is key to interpreting the results obtained from a se-ries of measurements. Other considerations also affect tech-nique selection. Table II summarizes some additional pointsto consider when matching an analytical requirement to a head-space oxygen–analysis tool. The table shows that analysis timesfor the five techniques range from minutes (GC, fluorescencequenching, quadrupole MS) to seconds (electrochemical meth-ods, FMS). All the instruments are available commercially, al-though at various stages of development, and vary in expensefrom $2,000 to $50,000. FMS and quadrupole MS are the leastcommercially developed of the techniques at this time, andmore work is needed before their routine use will be possible.Ease of calibration and use, which is a highly subjective cate-gorization, reflects the authors’ judgments about the relativeease of training others to operate the equipment. Other fac-tors such as versatility, portability, and suitability for use in amanufacturing setting also are important when one is choos-ing an instrument. For example, both headspace GC andquadrupole MS have numerous uses beyond oxygen monitor-ing, and one may want to acquire other information simulta-neously (e.g., amount of moisture, carbon dioxide, or othervolatiles). FMS is the only nondestructive technique of whichthe authors currently are aware, and it can be used only withpackage types that allow adequate transmittance of the instru-ment probe beam.

Package types. Identifying the type of package from whichoxygen levels must be sampled and analyzed is another con-sideration for determining the appropriate analysis tool. Mak-ing a decision from this angle requires information from bothTables I and II, especially the sample-volume requirements foreach technique that are listed in the latter. For the sake of brevity,only the three most common pharmaceutical packaging con-figurations (plastic bottles, glass vials–ampuls, and blister pack-ages) are discussed.

Plastic bottles with foil-induction inner seal. Plastic (e.g., HDPE) bot-tles with foil-induction inner seals (the most common have �33-, 75-, 120-mL volumes) are used commonly to packagepharmaceutical products. For the rapid and accurate determi-nation of headspace oxygen in a package of this type, all thetechniques discussed in this report apply, with the exceptionof FMS. Based on the authors’ experience, the electrochemicalmethods are best suited for large-volume (2 mL) packages,

and they display an unparalleled ease of use. These instrumentsalso tend to be among the most rugged of all the instrumen-tation described herein and seem to be the most well suited foruse by operators with a limited amount of technical training.

Glass vials and ampuls. Glass vials and ampuls are another com-monly used pharmaceutical packaging form, especially for paren-teral formulations. Any of the five techniques discussed in thisreport can be used to analyze headspace oxygen levels as longas sufficient volume is available. FMS offers considerable advan-tages for this package type because it is nondestructive. In ad-dition, FMS can be used for vials filled with a variety of media,including powders that may coat the walls of the container. Aslong as a portion of the analysis beam is transmitted, the dif-ferential absorptive signal processing inherent to the techniquecan be used to yield oxygen concentration values.

Blister packages. The small volume of individual blister cavitiesmakes this type of packaging the most challenging to analyzefor headspace oxygen levels. The challenges are twofold: the ex-tremely small volume available for sampling as well as sampleremoval or introduction without contamination. All the instru-ments that are applicable for small-volume measurements (i.e.,headspace GC, fluorescence quenching, quadrupole MS) havedrawbacks from a sampling perspective. Although future de-velopment in this area is needed, headspace GC seems to bemost suitable at this time because self-adhesive septa can beplaced on the package backing and a small portion of samplecan be withdrawn manually for injection. Other viable ap-proaches, including nondestructive techniques, currently arebeing investigated.

SummaryThis article has provided an introduction to the principles offive measurement techniques for analyzing the headspace oxy-gen levels in common types of pharmaceutical packages. Vari-ous performance criteria were applied to examine the fit of eachparticular tool to typical pharmaceutical package types. Futureresearch about the topic is needed to better address the ro-bustness of sample introduction techniques for small-volumesamples and perhaps to develop sensor technologies that wouldallow oxygen levels to be remotely sampled and profiled for aperiod of time from a small device integrated or inserted intothe package.

Table II: Some attributes of various headspace oxygen–sampling tools.Analysis Ease of Instrument Required

Instrument Time Calibration/Use* Availability Expense Sample Volume DestructiveGC Minutes 3 Commercial $40K� 10–30 �L YesElectrochemical Seconds 1 Commercial $4–10K 1–3 mL YesFluorescencequenching Minutes 3 Commercial $2–5K None** YesFMS Seconds 2 Semicommercial $35–50K None† NoQuadrupole MS Minutes 3 Semicommercial $25–50K 10–30 �L Yes

* 1 easiest to calibrate and use; 2 more difficult to calibrate and use; 3 most difficult to calibrate and use.** A sufficient sample volume must be available so that a probe can be inserted and make contact with the

sample, estimated at �100 �L.† Instrument is nondestructive and can be used on vials through which a laser can be transmitted.

7. J.N. Demas, B.A. DeGraff, and P.A. Coleman, “Oxygen Sensors Basedon Luminescence Quenching,”Anal. Chem. 71 (23), 793–801 (1999).

8. (a) M.J. Drinkwine and D. Lichtman, “Quadrupole Mass Analyzers,”in Partial-Pressure Analyzers and Analysis, N.R. Whetten and R. Long,Jr., Eds. (American Vacuum Society, New York, NY, 1978), pp. 235–278.(b) R.J. Ferran and S. Boumsellek, “High-Pressure Effects in Minia-ture Arrays of Quadrupole Analyzers for Residual Gas Analysis from10�9–10�2 Torr,” S. J.Vac. Sci. Technol. A 14, 1258–1265 (1996). PT

Pharmaceutical Technology JULY 2002 61

AcknowledgmentsThe authors thank the instrumentation vendors whose coop-eration greatly facilitated the work described in this article.Particularly, special thanks goes to Jim Veale and Bobby An-derson of Lighthouse Instruments, Inc. The authors also thankDenise Farquharson for assistance with the preparation of gasstandards.

DisclaimerThe information described in this report does not constituteendorsement by Merck & Co., Inc. of any particular instrumenttype or supplier.

References1. D.M. Johnson and L.C. Gu, “Autoxidation and Antioxidants,” in The

Encyclopedia of Pharmaceutical Technology, Vol. 1, J. Swarbric and J.C.Boylan, Eds. (Marcel Dekker, New York, NY, 1988), pp. 415–450.

2. K.A. Connors, G.L. Amidon, and L. Kennon, Chemical Stability of Phar-maceuticals (Wiley & Sons, New York, NY, 1979), pp. 80–98.

3. B. Kolb and L.S. Ettre, Static Headspace Gas Chromatography: Theoryand Practice (Wiley & Sons, New York, NY, 1997).

4. CRC Handbook of Chemistry and Physics, D.R. Lide, Ed. (CRC Press,Boca Raton, FL, 72d ed., 1991), p 2040.

5. J. Wang, Analytical Electrochemistry (Wiley & Sons, New York, NY,2000).

6. (a) G.C. Bjorklund, Opt. Lett. 5 (1), 15–17 (1980). (b) G.R. Janik, C.B.Carlisle, and T. F. Gallagher, J.Opt. Soc. Am. B 3 (8), 1070–1074 (1986).(c) J.A. Silver, Appl. Opt. 31 (6), 707–717 (1992). (d) D.E. Cooper andR.U. Martinelli, Laser Focus World 28 (11), 133–146 (November 1992).

Postdoctoral fellowshipsThe College of Pharmacy at Rutgers offers postdoctoral fellowshipsin partnership with various pharmaceutical companies.

The fellowships include practical industry experience; an adjunctfaculty appointment at the College of Pharmacy; and benefits such asa stipend, health insurance, and vacation and sick days. Applicantsmust possess a PharmD from an American Council onPharmaceutical Education–accredited college of pharmacy and mustsubmit a curriculum vitae, a written career-objective statement, andthree letters of recommendation.

For more information or to apply, contact Joseph A.Barone, PhD,Rutgers,The State University of New Jersey,College of Pharmacy,Department of Pharmacy Practice and Administration, Attention:Fellowship Program, 160 Frelinghuysen Rd., Piscataway, NJ 08854-8020, tel. 732.445.2675 ext. 600, fax 732.445.5767,http://pharmacy.rutgers.edu.

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Rapid Headspace Oxygen Analysis